Means and methods for producing neisseria meningitidis capsular polysaccharides of low dispersity

ABSTRACT

The present invention relates to in vitro methods for producing  Neisseria meningitidis  capsular polysaccharides which have a defined length. The present invention also relates to compositions comprising at least one capsule polymerase, at least one donor carbohydrate and at least one acceptor carbohydrate, wherein the ratio of donor carbohydrate to acceptor carbohydrate is a ratio from 10:1 to 400:1. Moreover, the present invention provides truncated versions of the capsule polymerases of  Neisseria meningitidis  serogroups A and X. Also provided herein are pharmaceuticals, in particular vaccines, comprising the synthetic capsular polysaccharides of  Neisseria meningitidis  which have a defined length. Furthermore, the invention provides for methods for the production of said vaccines.

The present invention relates to in vitro methods for producingNeisseria meningitidis capsular polysaccharides which have a definedlength. The present invention also relates to compositions comprising atleast one capsule polymerase, at least one donor carbohydrate and atleast one acceptor carbohydrate, wherein the ratio of donor carbohydrateto acceptor carbohydrate is a ratio from 10:1 to 400:1. Moreover, thepresent invention provides truncated versions of the capsule polymerasesof Neisseria meningitidis serogroups A and X. Also provided herein arepharmaceuticals, in particular vaccines, comprising the syntheticcapsular polysaccharides of Neisseria meningitidis which have a definedlength. Furthermore, the invention provides for methods for theproduction of said vaccines.

Bacterial meningitidis is a serious threat to global health. It isestimated that each year bacterial meningitidis accounts for 170,000deaths worldwide (WHO, http://www.who.int/nuvi/meningitidis/en/).Despite the availability of potent antimicrobial agents, case-fatalityrates are high (10-40%) and survivors frequently suffer from sequelaesuch as neurologic disability or limb loss and deafness (Van Deuren etal., Clin Micriobiol Rev 2000; 13(1): 144-166; Kaper et al., Nat RevMicrobiol 2004, 2(2): 123-140). Neisseria meningitidis (Nm) is one ofthe most important causative agents of bacterial meningitidis because ofits potential to spread in epidemic waves (Kaper et al., Nat RevMicrobiol 2004, 2(2): 123-140; Rosenstein et al., N Eng J Med 2001,344(18): 1378-1388). Crucial virulence determinants of disease causingNm species are their extracellular polysaccharide capsules that areessential for meningococcal survival in human serum (Vogel et al.,Infect Immun 1997, 65(10): 4022-4029). Based on antigenic variation ofthese polysaccharides at least twelve different serogroups of Nm havebeen identified (A, B, C, E29, H, I, K, L, W-135, X, Y and Z), but onlysix (A, B, C, W-135, Y and X) account for virtually all cases ofdisease; see also Frosch, M., VOGEL, U. (2006) “Structure and geneticsof the meningococcal capsule.” In Handbook of Meningococcal Disease.Frosch, M., Maiden, M. C. J. (eds). Weinheim: Wiley-VCH.

Serogroup A (NmA) and C (NmC) are the main causes of meningococcalmeningitidis in sub-Saharan Africa, while serogroups B (NmB) and C arethe major disease causing isolates in industrialized countries. However,serogroups W-135 (NmW-135) and Y (NmY) are becoming increasinglyprevalent. For NmW-135, this is most explicitly evidenced by the 2002epidemic in Burkina Faso with over 13,000 cases and more than 1,400deaths (Connolly et al., Lancet 2004, 364(9449): 1974-1983; WHO,Epidemic and Pandemic Alert and Response (EPR) 2008). In contrast, NmYis gaining importance in the United States where its prevalenceincreased from 2% during 1989-1991 to 37% during 1997-2002 (Pollard etal., J Paediatr Child Health 2001, 37(5): S20-S27). However, also thepreviously only sporadically found serogroup X (NmX) appeared with highincidence in Niger and caused outbreaks in Kenya and Uganda (Biosier etal., Clin Infect Dis 2007, 44(5): 657-663; Lewis, WHO Health Action inCrisis 1, 6 2006).

The serogroups A, B, C, 29E, H, I, K, L, W-135, X, Y and Z are wellknown in the art and are described, e.g., in Frosch, M., VOGEL, U.(2006) loc. cit. The capsular polysaccharides (CPS) of all serogroupsare negatively charged linear polymers. Serogroup B and C are encapsuledin homoplymeric CPS composed of sialic acid (Neu5Ac) moieties that arelinked by either α-2-8 glycosidic linkages in serogroup B or by α-2-9linkages in serogroup C (Bhattacharjee et al., J Biol Chem 1975, 250(5):1926-1932). Serogroup W-135 and Y both are heteropolymers. They arecomposed of either galactose/Neu5Ac repeating units[→6)-α-D-Glcp-(1→4)-α-Neu5Ac-(2→]_(n) in serogroup W-135 orglucose/Neu5Ac repeating units [→6)-α-D-Galp-(1→4)-α-Neu5Ac-(2→]_(n) inserogroup Y (Bhattacharjee et al., Can J Biochem 1976, 54(1): 1-8). TheCPS of NmA and NmX do not contain Neu5Ac moieties, but are instead builtfrom N-Acetyl-mannosamine 1-phosphate [→6)-α-D-ManpNAc-(1→OPO₃→]_(n) orN-Acetyl-glucosamine 1-phosphate [→6)-α-D-GlcpNAc-(1→OPO₃→]_(n)repeating units, respectively (Bundle et al., Carbohydr Res 1973, 26(1):268-270; Bundle et al., J Biol Chem 1974, 249(15): 4797-4801); Bundle etal., J Biol Chem 1974, 249(7): 2275-2281; Jennings et al., J Infect Dis1977, 136 Suppl: S78-S83).

The CPS of disease causing Nm are attractive vaccine candidates andpolysaccharide or polysaccharide-conjugate vaccines are available forserogroups A, C, Y, and W-135 (Broker et al., Minerva Med 2007,98(5):575-589). Recently, also vaccines for serogroup X have beendescribed (Micoli, P. Proc Natl Acad Sci USA. (2013) 110: 19077-82).

Key enzymes in the CPS biosynthesis are membrane associated capsulepolymerases. Candidate genes have been identified for all six diseasecausing serogroups (Frosch et al., Proc Natl Acad Sci USA 1989, 86(5):1669-1673; Claus et al., Mol Gen Genet 1997, 257(1): 28-34; Tzeng etal., Infect Immun 2003, 71(2): 6712-6720). However, our knowledge ofenzymology or structure-function relations of those important enzymes isstill very limited.

Until now, polysaccharide production for neisserial vaccines stillrequires fermentation of Neisseria meningitidis with subsequentmultistep purification of the polysaccharides from the culture medium.In these conventional vaccine production processes, the obtained capsulepolysaccharides are mostly long polymers. However, for vaccineproduction, the oligosaccharides ranging in size between degree ofpolymerization (DP) DP10 to DP60 are needed. Therefore, afterpurification of the capsule polysaccharides from the culture medium, theobtained polysaccharides have to be fragmented and the fragments have tobe characterized and sorted by length. Foremost the biohazard associatedwith mass production of pathogenic Neisseria stains, but also thetechnological platforms needed to produce homogenous oligosaccharidefractions for vaccine production and their functionalization forcoupling to carrier proteins (e.g. an inactive form of Diphteria-toxinor tetanus toxoid), make the conventional vaccine production processescost intensive and time consuming. Moreover, due to the complexity ofthe vaccine production chain it can hardly be carried out in developingcountries where most of the Nm infections occur. In addition, theseconventional vaccine production processes are at risk for contaminationsby neisserial toxins, media components or chemicals required forsubsequent purification procedures.

Thus, the technical problem underlying the present invention is theprovision of means and methods which lead to a more convenientproduction process of vaccines against Neisseria meningitidis.

The technical problem has been overcome by the methods of the presentinvention for producing capsular polysaccharides of Neisseriameningitidis in vitro which have a defined length and carry functionalgroups at the reducing end for carrier protein coupling as will bedetailed below. In particular, the herein provided means and methodslead to a vaccine production process which is less expensive and lesstime consuming as compared to the processes of the prior art.Furthermore, these convenient processes as provided herein can also becarried out in the developing world. Accordingly, the technical problemis solved by the provision of the embodiments as defined in the claims.

Accordingly, the present invention relates to an in vitro method forproducing Neisseria meningitidis capsular polysaccharides which have adefined length, said method comprising the steps:

-   (a) incubating at least one capsule polymerase with at least one    donor carbohydrate and at least one acceptor carbohydrate; wherein    the ratio of donor carbohydrate to acceptor carbohydrate is a ratio    from 10:1 to 400:1; and-   (b) isolating the resulting capsular polysaccharides,    wherein the capsule polymerase is the capsule polymerase of    Neisseria meningitidis serogroup A or a truncated version of the    capsule polymerase of Neisseria meningitidis serogroup X.

The present invention solves the above identified technical problemsince, as documented herein below and in the appended Examples, it wassurprisingly found that capsule polysaccharides of Neisseriameningitidis (Nm) serogroups A and X which have a uniform and definedlength can be produced in vitro.

The inventive means and methods have the advantageous property that thebiohazard in association with large scale Nm cultures and thesignificant coasts for purifying capsular polysaccharides which have thedesired length can be avoided. Moreover, the inventive methods providethe perspective that vaccine production chains can be established alsoin areas that are lacking advanced infrastructural conditions. Inparticular, the present invention provides for a pyrogen-free, enzymecatalyzed in vitro synthesis production of capsular polysaccharideswhich have a defined length.

More specifically, the inventors of the present invention surprisinglyidentified that the capsular polymerase (CP) of Nm serogroup A (CP-A)can be regulated by the ratio of donor to acceptor saccharides. Morespecifically, in context of the present invention it has been found thatthis capsular polymerase can be regulated via the donor-acceptor ratiosuch that it produces capsular polysaccharides with a uniform anddefined length. This finding is of high relevance as for vaccineproduction, neisserial capsular polysaccharides with a uniform anddefined length are required. In addition, regulation of the capsulepolymerases of Nm serogroup A by the donor-acceptor ratio is highlyunexpected as the full length capsule polymerases of Nm serogroups X, Yand W-135 cannot be easily regulated by donor-acceptor ratio.

Furthermore, the inventors of the present invention astonishingly foundthat, when terminally truncated, also the capsule polymerase of Nmserogroup X (CP-X) can be regulated via the donor-acceptor ratio inorder to produce capsular polysaccharides with a uniform and definedlength. Moreover, as shown in the illustrating appended examples, thisterminally truncated version of the capsule polymerase of Nm serogroup Xhas the additional advantage that it can also be regulated by thereaction time. Thus, as shown in the appended examples, by choosing asuitable reacting time (e.g. 3 to 45 minutes), the terminally truncatedversion of the capsule polymerase of Nm serogroup X can be forced toproduce capsular polysaccharides with a uniform and defined length ofDP10 to DP60. This length is needed for effective vaccine production. Itis noted that the inventive truncated (e.g. terminally truncated)capsular polymerase of NmX has the additional advantage that it has asignificantly improved activity as compared to its full lengthcounterpart. This is a surprising finding as the prior art has shownthat truncated versions of the capsule polymerase of Nm serogroup B(CP-B) have the same or even less activity as compared to the fulllength CP-B (Keys, Analytical Biochemistry 427, 60-68 (2012)).

Thus, the present invention paves the way for efficient, economical andfast in vitro production methods for Nm capsule polysaccharides, whichare suitable for vaccine development. Furthermore, in context of thepresent invention two important features of the capsule polymerase ofNmA (also called CP-A or CsaB) have been identified: (i) the minimalefficient acceptor is the dimer and (ii) the reducing end phosphategroup can be extended with rather large chemical groups (such asdecyl-ester groups). This latter finding bears the perspective thatchain elongation can be primed with reagents of very high purity andfunctional groups that facilitate conjugation of glycans to carrierproteins in the vaccine production chain.

Moreover, in context of the present invention it was further found thatan O-acetyltransferase (e.g. the CsaC of NmA) can be applied in theinventive in vitro production methods and directly effectingO-acetylation of the produced capsular polysaccharides. This is ofadvantage as usually for vaccine production Nm capsular polysaccharidesmust be O-acetylated. In addition, O-acetylation has also an effect onthe immunogenicity of the produced capsular polysaccharides. Naturalcapsule polysaccharides of NmA are O-acetylated in position 3-O and to aminor extend in position 4-O of ManNAc (Gudlavalleti, Carbohydr. Res.(2006) 341: 557-562). It has been shown that capsular polysaccharides ofNmA are immunogenic only if they are O-acetylated (Berry et al., 2002Infection and immunity 70 (7), 3707-3713). As demonstrated in theappended examples, during in vitro synthesis of CPS anO-acetyltransferase (such as the CsaC of NmA) can be directly added tothe mixture comprising the capsule polymerase, the donor carbohydrate(s)and the acceptor carbohydrate(s). Thus, in vitro production andO-acetylation can be performed in a one-pot reaction. Accordingly, theO-acetyltransferase may be used in step (a) of the herein described invitro method for producing Nm capsular polysaccharides. In addition oralternatively, O-acetylation may be performed as an additional stepafter the synthesis of the Nm capsular polysaccharides. Thus, in theherein described in vitro method for producing Nm capsularpolysaccharides, O-acetylation may be performed after step (a) in theadditional step (a′).

Accordingly, the present invention provides for effective in vitromethods for producing exactly the type of Nm capsular polysaccharideswhich is required for vaccine production (i.e. capsular polysaccharideswhich have a defined length and which are O-acetylated). Thereby, thepreviously used cost- and time-intensive vaccine production processescan be avoided.

In vitro methods for producing synthetic capsular polysaccharides ofNeisseria meningitidis have been described in the prior art (WO2011/023764 A1). However, the prior art does not disclose how togenerate synthetic Nm capsular polysaccharides which have a uniform anddefined length and the prior art does not disclose that anO-acetyltransferase can be applied in the in vitro production ofcapsular polysaccharides to obtain O-acetylated capsularpolysaccharides.

By contrast, in the methods of the present invention, isolated Nmcapsular polymerases are regulated by the donor-acceptor ratio and/or bythe reaction time to allow for the production of Nm capsularpolysaccharides which have a uniform and defined length. Accordingly,the invention provides for methods for producing Nm capsularpolysaccharides, wherein the capsular polymerases are regulated by thedonor-acceptor ratio and/or the reaction time so as to produce capsularpolysaccharides which have a uniform and defined length.

Moreover, a further advantageous property of the methods of theinvention is that they can be combined with an O-acetyltransferase togenerate O-acetylated capsular polysaccharides which can directly beused for vaccine production.

Thus, the present invention relates to an in vitro method for producingNeisseria meningitidis capsular polysaccharides which have a definedlength, said method comprising the steps:

-   (a) incubating at least one capsule polymerase with at least one    donor carbohydrate and at least one acceptor carbohydrate; wherein    the ratio of donor carbohydrate to acceptor carbohydrate is a ratio    from about 10:1 to about 400:1; and-   (b) isolating the resulting capsular polysaccharides,    wherein the capsule polymerase is the capsule polymerase of    Neisseria meningitidis serogroup A or a truncated version of the    capsule polymerase of Neisseria meningitidis serogroup X, and    wherein in step (a) the corresponding capsular polysaccharide is    produced. Said production method is an in vitro production method    and allows for isolation of capsular polysaccharides from the    incubation mixture as defined in step (a); i.e. this isolation    allows for the separation of the produced capsular polysaccharides    from the capsule polymerase(s), the donor carbohydrate(s) and/or the    acceptor carbohydrate(s).

The appended examples demonstrate that capsular polysaccharides whichhave the desired length of DP10 to DP60 can be produced by using CP-A ora terminally truncated version of CP-X and a ratio of donor carbohydrateto acceptor carbohydrate which is approximately between 10:1 and 400:1.Therefore, in the herein described methods, if the length of the Nmcapsular polysaccharides is regulated via the ratio of donorcarbohydrate to acceptor carbohydrate, than the donor:acceptor ratio is10:1 to 400:1 (e.g. 20:1 to 400:1). For example, the ratio may be 10:1,20:1, 40:1, 60:1, 80:1, 100:1, 150:1, 200:1, 220:1, 240:1, 250:1, 300:1,350:1 or 400:1. In particular aspects of the invention also other donoracceptor ratios, e.g. ratios from 2:1 to 2000:1 may be used.

If CP-A is used in the in vitro methods described herein, than the ratioof donor carbohydrate to acceptor carbohydrate is preferably from 10:1to 240:1, and more preferably from 10:1 to 80:1, and even morepreferably from 10:1 to 75:1, and even more preferably from 20:1 to75:1, and most preferably from 20:1 to 60:1 (e.g. 40:1 to 60:1 or 50:1).The term “CP-A” includes a polypeptide which has the amino acid sequenceof SEQ ID NO: 9 or 10 and a polypeptide which has at least 80%(preferably at least 90%) sequence identity to SEQ ID NO: 9 (CP-Awildtyp/full-length) or to SEQ ID NO: 10 (ΔN69-CP-A).

If the terminally truncated CP-X is used and if it is regulated via theratio of donor carbohydrate to acceptor carbohydrate (and not via thereaction time) then the following ratios are preferably used. Forexample, if the capsule polymerase is a polypeptide which has the aminoacid sequence of SEQ ID NO: 28 or which has at least 80% (preferably atleast 90%) sequence identity to SEQ ID NO: 28 (ΔC99-CP-X), then theratio of donor carbohydrate to acceptor carbohydrate is preferably from10:1 to 80:1, more preferably from 20:1 to 60:1, even more preferablyfrom 20:1 to 50:1, and most preferably from 33:1 to 50:1. In addition,if the capsule polymerase is a polypeptide which has the amino acidsequence of SEQ ID NO: 25 or which has at least 80% (preferably at least90%) sequence identity to SEQ ID NO: 25 (ΔN58-CP-X), then the ratio ofdonor carbohydrate to acceptor carbohydrate is preferably from 10:1 to80:1, more preferably from 20:1 to 50:1, and most preferably from 20:1to 33:1 (e.g. 33:1). Furthermore, if the capsule polymerase is apolypeptide which has the amino acid sequence of SEQ ID NO: 29 or whichhas at least 80% (preferably at least 90%) sequence identity to SEQ IDNO: 29 (ΔN58ΔC99-CP-X), then the ratio of donor carbohydrate to acceptorcarbohydrate is preferably from 50:1 to 400:1, more preferably from 80:1to 400:1, even more preferably from 100:1 to 400:1, and most preferablyfrom 100:1 to 200:1.

All capsule polymerases which are applied in the herein describedmethods are functional. For example, a polypeptide which has at least80% (preferably at least 90%) sequence identity to any one of thesequences of SEQ ID NOs: 9, 10, 25, 28, and 29 is considered to befunctional, i.e. to have the same function as the polypeptide which hasthe sequence of SEQ ID NO: 9, 10, 25, 28, or 29, respectively. Inparticular, the function of a polypeptide having the sequence of SEQ IDNO: 9 or 10 is the ability to transfer ManNAc-1P (or a derivativethereof) from UDP-ManNAc (or a derivative thereof) onto an acceptorcarbohydrate. Preferably, at least 80% of the acceptor carbohydrates arepolysaccharides with a DP of ≧4. In addition, the function of apolypeptide having the sequence of any one of SEQ ID NOs: 25, 28, and 29is the ability to transfer GlcNAc-1P (or a derivative thereof) fromUDP-GlcNAc (or a derivative thereof) onto an acceptor carbohydrate.

If (in the herein described in vitro methods for producing Nm capsularpolysaccharides (Nm CPS) which have a defined length) the length of theproduced capsular polysaccharides is regulated via the donor:acceptorratio, then 50-100 nM of the capsular polymerase may be used. The amountof donor carbohydrate may be 1-100 mM, e.g. 5-10 mM. The amount ofacceptor carbohydrate is adjusted to the amount of donor carbohydrate soas to result in the desired donor:acceptor ratio ratio. For example, theamount of acceptor carbohydrate may be 1-1000 μM, e.g. 10-40 μM.Preferably, at least 80% of the acceptor carbohydrates arepolysaccharides with a DP of ≧4.

For example, if in the herein described methods for producing Nmpolysaccharides with a defined length the CP-A is used, then 1-100 nM(preferably 70-100 nM) of CP-A, 10 mM donor carbohydrate (e.g. a dimerof ManNAc-1-phosphate), and an amount of acceptor carbohydrate (e.g.purified capsular polysaccharides of NmA) resulting in a ratio of donorcarbohydrate to acceptor carbohydrate which is from 10:1 to 400:1,preferably from 10:1 to 240:1, more preferably from 10:1 to 80:1, evenmore preferably from 10:1 to 75:1, even more preferably from 20:1 to75:1, and most preferably from 20:1 to 60:1 (e.g. 40:1 to 60:1 or 50:1)may be used. In a particular example of the herein described methods forproducing Nm polysaccharides with a defined length, 70-100 nM of CP-A,10 mM donor carbohydrate (e.g. UDP-ManNAc; or UDP-GlcNAc, wherein instep (a) the capsule polymerase is further incubated with theUDP-GlcNAc-epimerase), and an amount of acceptor carbohydrate (e.g.purified capsular polysaccharides of NmA of which at least 80% have a DPof ≧4) resulting in a ratio of donor carbohydrate to acceptorcarbohydrate which is from 20:1 to 60:1 is used. The term “CP-A”includes a polypeptide having the amino acid sequence as shown in SEQ IDNO: 9 (CP-A wildtyp/full-length) or SEQ ID NO: 10 (ΔN69-CP-A) and apolypeptide with an amino acid sequence having at least 80% (preferablyat least 90%) sequence identity to SEQ ID NO: 9 or to SEQ ID NO: 10 andbeing functional.

In another particular example of the herein described methods, thelength of the produced Nm polysaccharides is regulated via thedonor:acceptor ratio and 50-70 nM of a polypeptide having the amino acidsequence of SEQ ID NO: 25 (ΔN58-CP-X), 28 (ΔC99-CP-X), or 29(ΔN58ΔC99-CP-X), or having an amino acid sequence which is at least 80%(preferably at least 90%) identical to SEQ ID NO: 25, 28, or 29 andbeing functional; 10 mM donor carbohydrate (e.g. UDP-GlcNAc), and anamount of acceptor carbohydrate (e.g. purified capsular polysaccharidesof NmX of which at least 80% have a DP of ≧4) resulting in the desiredratio of donor carbohydrate to acceptor carbohydrate is used.

In another example of the invention, the length of the Nm CPS isregulated by the reaction time, and 50-70 nM capsular polymerase (e.g.ΔC99-CP-X or ΔN58ΔC99-CP-X), 10 mM donor carbohydrate (e.g. UDP-GlcNAc)and 10-30 μM acceptor carbohydrate (e.g. purified capsularpolysaccharides of NmX of which at least 80% have a DP of ≧4) is used.

Step (a) of the herein provided in vitro method may be performed byincubating the capsule polymerase, the donor carbohydrate and theacceptor carbohydrate in a reaction buffer (e.g. a buffer comprising 50mM Tris pH 8.0 and 20 mM MgCl₂). The incubation in step (a) may beperformed at a temperature range between 20° C. and 40° C., preferablybetween 25° C. and 37° C., more preferably between 30 and 37° C.

In context of the present invention it has surprisingly and unexpectedlybeen found that in the herein described methods, if the length of the Nmcapsular polysaccharides is regulated via the ratio of donorcarbohydrate to acceptor carbohydrate (and not via the reaction time)and if the CP-A or a C-terminally truncated version of the CP-X is used(e.g. ΔC99-CP-X), then the donor:acceptor ratio (i.e. the donor:acceptorquotient) reflects the avDP (i.e. the length of the major species withinthe produced capsular polysaccharides). For example, if thedonor:acceptor ratio is 15:1, then the CP-A or the C-terminallytruncated CP-X produces capsular polysaccharides which have an avDP15.Or, if the donor:acceptor ratio is 50:1, then the CP-A or theC-terminally truncated CP-X produces capsular polysaccharides which havean avDP50 (see, e.g., FIG. 1A, 2C, 2F). Or, in other words, if thedonor:acceptor ratio is 15:1 (or 50:1, respectively), then the majorspecies (i.e. the main components) of the produced capsularpolysaccharides have a length around DP15 (or DP50, respectively). Or,in other words, the product pool has an avDP15 (or avDP50, respectively)and is narrowly distributed around the major species having a DP of 15(or 50, respectively). In line with this, if the donor:acceptor ratio isfrom 15:1 to 20:1, then the major species of the produced capsularpolysaccharides have a length of avDP15 to avDP20.

Accordingly, the present invention relates to an in vitro method forproducing Neisseria meningitidis capsular polysaccharides which have adefined length, said method comprising the steps:

-   (a1) incubating at least one capsule polymerase with at least one    donor carbohydrate and at least one acceptor carbohydrate; and    thereby producing capsule polysaccharides, wherein at least 60%    (preferably at least 80% and most preferably at least 90%) of the    produced capsular polysaccharides have a length or in other words a    degree of polymerisation corresponding to the ratio of donor    carbohydrate to acceptor carbohydrate −20/+30 repeating units; and-   (b) isolating the resulting capsular polysaccharides,    wherein the capsule polymerase is the CP-A or a C-terminally    truncated version of the CP-X. The C-terminally truncated version of    the CP-X may be a polypeptide which comprises:    -   (i) an amino acid sequence encoded by a nucleic acid molecule        which comprises the nucleic acid sequence of SEQ ID NO: 20        (ΔC99-CP-X); or a nucleic acid sequence having at least 80%        (preferably at least 90%) identity to the nucleic acid sequence        of SEQ ID NO: 20 and encoding a functional polypeptide; wherein        the function comprises the ability to transfer GlcNAc-1P from        UDP-GlcNAc onto an acceptor carbohydrate; or    -   (ii) the amino acid sequence of SEQ ID NO: 28 (ΔC99-CP-X); or an        amino acid sequence having at least 80% (preferably at least        90%) identity to SEQ ID NO: 28 and being functional, wherein the        function comprises the ability to transfer GlcNAc-1P from        UDP-GlcNAc onto an acceptor carbohydrate.

As indicated, in the above described method, the produced capsularpolysaccharides comprise as a main component capsular polysaccharideshaving a DP corresponding to about the ratio of donor carbohydrate toacceptor carbohydrate. Preferably, at least 80% of the acceptorcarbohydrates are polysaccharides with a DP of ≧4. Thus, the length ofthe produced capsular polysaccharides can easily be regulated via thedonor:acceptor ratio. One aspect of the invention relates to the abovedescribed method, wherein the donor:acceptor ratio is from 15:1 to 20:1,and wherein at least 60% (preferably 80%, most preferably 90%) of theproduced capsular polysaccharides have a length, or in other words, adegree of polymerisation corresponding to the ratio of donorcarbohydrate to acceptor carbohydrate −20/+30 repeating units.

Herein the term “Neisseria meningitidis capsular polysaccharides whichhave a defined length” means that the produced capsular polysaccharideshave a defined DP (degree of polymerization) or a defined avDP (averagedegree of polymerization), e.g. a DP between 10 and 60 or an avDPbetween 15 and 20. The term “Neisseria meningitidis capsularpolysaccharides which have a defined length” preferably means that atleast 60% (more preferably at least 80%, most preferably at least 90%)of the produced capsular polysaccharides have a length between DP10 andDP60. For example, the DP may be between DP20 and DP60. The DP indicatesthe number of monosaccharide-units which built up the capsularpolysaccharide. For example, a DP of 10 means that the capsularpolysaccharide consists of 10 monosaccharide-units and a DP of 60 meansthat the capsular polysaccharide consists of 60 monosaccharide-units.Accordingly, a NmX capsular polysaccharide having DP10 means that thecapsular polysaccharide consists of 10 GlcNAc-1P repeating units. Acidichydrolysis used to produce oligosaccharide fragments form longpolysaccharide chains, results in fragments of different lengths, whichin subsequent chromatographic steps can be separated into size classes.The average DP (avDP) describes the dispersion of chains with one classand can be calculated according to established protocols as described,e.g. in Berti (2012) Vaccine 30 (45), 6409-6415. Or, in other words, theavDP describes the average length of a polysaccharide pool. Alternativeways to determine the DP of oligosaccharides are high percentagepolyacrylamide gel electrophoresis or HPLC-anion-exchange chromatographyas demonstrated in the appended examples. The avDP may be determined bythe protocol described in Berti (2012) 30(45): 6409-15., which includes³¹P NMR and High Performance Anionic Exchange Chromatography with PulsedAmperometric Detection (HPAEC-PAD). For vaccine production usuallycapsular polysaccharides having an avDP of 15 to 20 is used. The productpool of polysaccharides with an avDP of 15 to 20 consists ofpolysaccharides having a DP of 10 to 60. The herein provided in vitromethods have the advantage that capsule polysaccharides with the lengthwhich is desired for vaccine production (i.e. DP10 to DP60) can directlybe produced. Herein the term “Neisseria meningitidis capsularpolysaccharides which have a defined length” also means that theproduced capsular polysaccharides have a low dispersity (i.e. a narrowproduct distribution).

In the above described in vitro method, if the capsule polymerase of Nmserogroup A is used, than capsule polysaccharides of Nm serogroup A areproduced. Analogously, if a truncated version of the capsule polymeraseof Nm serogroup X is used, that capsule polysaccharides of Nm serogroupX are produced.

As described herein and illustrated in the appended examples, one aspectof the present invention relates to the identification of terminallytruncated versions of the capsule polymerase of Nm serogroup X (CP-X,also designated as CsxA). These terminally truncated proteins have theadvantage that they have an increased activity as compared to the fulllength CP-X. A further advantageous property of these terminallytruncated proteins is that they can be regulated by the ratio of donorcarbohydrates to acceptor carbohydrates such that they produce capsularpolysaccharides which have a uniform and defined length. This issurprising since the full length CP-X cannot be regulated by thedonor-acceptor ratio. Furthermore, in context of the present inventionit has astonishingly been found that these terminally truncated versionsof the CP-X can also be regulated by the reaction time so as to producecapsular polysaccharides which have a uniform and defined length. Morespecifically, the time how long the terminally truncated CP-X isincubated with the donor carbohydrates (and, optionally, the acceptorcarbohydrates) regulates the length of the produced capsulepolysaccharides. Accordingly, an incubation time can be chosen whichresults in the production of capsule polysaccharides of NmX which havethe length which is desired for vaccine production (i.e. DP10 to DP60).Accordingly, one embodiment of the invention relates to an in vitromethod for producing Neisseria meningitidis capsular polysaccharideswhich have a defined length, said method comprising the steps:

-   (a) incubating at least one capsule polymerase with at least one    donor carbohydrate and at least one acceptor carbohydrate; wherein    the incubation time ranges from 3 to 45 minutes; and-   (b) isolating the resulting capsular polysaccharides,    wherein the capsule polymerase is a truncated version of the capsule    polymerase of Neisseria meningitidis serogroup X.

Or, in other words, the invention relates to an in vitro method forproducing Neisseria meningitidis capsular polysaccharides which have adefined length, said method comprising the steps:

-   (a) incubating at least one capsule polymerase with at least one    donor carbohydrate and at least one acceptor carbohydrate; wherein    the incubation time ranges from about 3 to about 45 minutes; and-   (b) isolating the resulting capsular polysaccharides,    wherein the capsule polymerase is a truncated version of the capsule    polymerase of Neisseria meningitidis serogroup X, and wherein in    step (a) the corresponding capsular polysaccharide is produced. Said    production method is an in vitro production method and allows for    isolation of capsular polysaccharides from the incubation mixture as    defined in step (a); i.e. this isolation allows for the separation    of the produced capsular polysaccharides from the capsule    polymerase(s), the donor carbohydrate(s) and/or the acceptor    carbohydrate(s).

Due to the distributive nature of the elongation mechanism used by thetruncated CP-X, the reaction can be stopped at any time whereby theresulting avDP depends on the reaction time. Therefore, if the truncatedversion of the CP-X is regulated via the reaction time, then the ratioof donor carbohydrate to acceptor carbohydrate is not relevant. Forexample, the ratio of donor carbohydrate to acceptor carbohydrate canrange from 1:1 to 50000:1, preferably from 10:1 to 10000:1 [Hier wurdeentsprechend der Ansprüche 10:1 to 10000:1 gewählt] (e.g. from 200:1 to10000:1, or more preferably from 100:1 to 8000:1).

In context of the present invention it has surprisingly been found thatthe herein described capsule polymerases are active even if they arecoupled to a solid phase. Immobilizing a component on a solid phase hasthe advantage that it reduces labor in the isolating step (b). Morespecifically, immobilizing a component to a solid phase omits furtherpurification steps to remove the produced capsular polysaccharides fromthe reaction mixture of step (a). Thus, in the herein provided in vitromethods for producing Nm capsular polysaccharides, one of the componentsmay be immobilized (i.e. bound/coupled) on a solid phase. For example,the donor carbohydrate or the capsule polymerase may be immobilized on asolid phase. The solid phase may be column, such as a His-Trap column(i.e. a column having His-Trap beads). In a particular aspect of theinventive in vitro methods, the capsule polymerase is immobilized on asolid phase. For example, a polypeptide which has the amino acidsequence of SEQ ID NO: 29 (ΔN58ΔC99-CP-X) or which has at least 80%(preferably at least 90%) sequence identity to SEQ ID NO: 29 and beingfunctional may be immobilized on a solid phase.

It is noted that the capsule polymerase of Nm serogroup X is capable ofa de novo synthesis of capsular polysaccharides. Or, in other words, theCP-X is able to synthesize capsular polysaccharides in the absence ofacceptor carbohydrates (Fiebig, Glycobiology (2014) 24:150-8). Thus, ifthe terminally truncated version of CP-X is regulated by the reactiontime, the presence of acceptor carbohydrates is optional. Accordingly,one aspect of the invention relates to an in vitro method for producingNm capsular polysaccharides of NmX which have a defined length, saidmethod comprising the steps:

-   (a) incubating a terminally truncated version of the CP-X with at    least one donor carbohydrate (and, optionally, with at least one    acceptor carbohydrate); wherein the incubation time ranges from 3 to    45 minutes; and-   (b) isolating the resulting capsular polysaccharides.

As described herein and illustrated in the appended examples, theinventive terminally truncated versions of the capsule polymerase of Nmserogroup X can be regulated by both, the donor-acceptor ratio and thereaction time (i.e. the time how long the terminally truncated versionof the CP-X is incubated with the donor saccharides and the acceptorsaccharides).

As mentioned above, if the truncated version of the CP-X is regulatedvia the reaction time, then the ratio of donor carbohydrate to acceptorcarbohydrate is not relevant. Thus, as also demonstrated in the appendedExamples, if the length of the Nm capsular polysaccharides is regulatedby the reaction time, then higher donor-acceptor ratios can be used (toproduce Neisseria meningitidis capsular polysaccharides which have adefined length) as compared to the method wherein the length of the Nmcapsular polysaccharides is regulated via the donor-acceptor ratio (andnot via the reaction time). Accordingly, one aspect of the inventionrelates to an in vitro method for producing Neisseria meningitidiscapsular polysaccharides which have a defined length, said methodcomprising the steps:

-   (a) incubating at least one capsule polymerase with at least one    donor carbohydrate and at least one acceptor carbohydrate; wherein    -   (i) the ratio of donor carbohydrate to acceptor carbohydrate is        a ratio from 10:1 to 10000:1 [Hier wurde entsprechend der        Ansprüche 10:1 to 10000:1 gewählt], and    -   (ii) the incubation time ranges from 3 to 45 minutes; and-   (b) isolating the resulting capsular polysaccharides,    wherein the capsule polymerase is a truncated version of the capsule    polymerase of Neisseria meningitidis serogroup X.

Or, in other words, the invention relates to an in vitro method forproducing Neisseria meningitidis capsular polysaccharides which have adefined length, said method comprising the steps:

-   (a) incubating at least one capsule polymerase with at least one    donor carbohydrate and at least one acceptor carbohydrate; wherein    -   (i) the ratio of donor carbohydrate to acceptor carbohydrate is        a ratio from about 10:1 to about 10000:1, and    -   (ii) the incubation time ranges from about 3 to about 45        minutes; and-   (b) isolating the resulting capsular polysaccharides,    wherein the capsule polymerase is a truncated version of the capsule    polymerase of Neisseria meningitidis serogroup X; and wherein in    step (a) the corresponding capsular polysaccharide is produced. Said    production method is an in vitro production method and allows for    isolation of capsular polysaccharides from the incubation mixture as    defined in step (a); i.e. this isolation allows for the separation    of the produced capsular polysaccharides from the capsule    polymerase(s), the donor carbohydrate(s) and/or the acceptor    carbohydrate(s).

Or, in other words, the invention relates to an in vitro method forproducing Nm capsular polysaccharides of NmX which have a definedlength, said method comprising the steps:

-   (a) incubating a terminally truncated version of CP-X with at least    one donor carbohydrate and at least one acceptor carbohydrate;    wherein    -   (i) the ratio of donor carbohydrate to acceptor carbohydrate is        a ratio from 10:1 to 10000:1, and    -   (ii) the incubation time ranges from 3 to 45 minutes; and-   (b) isolating the resulting capsular polysaccharides.

The appended examples demonstrate that capsular polysaccharides of NmXwhich have the desired length of DP10 to DP60 can be produced by using aterminally truncated version of CP-X and a ratio of donor carbohydrateto acceptor carbohydrate which is approximately between 10:1 and 400:1.The appended Examples also demonstrate that higher donor-acceptor ratios(e.g., 200:1 or 8000:1) can be used to produce Nm capsulepolysaccharides which have a defined length, if the terminally truncatedversion of the CP-X is regulated via the reaction time (i.e. if thereaction time is from 3 to 45 min).

Accordingly, if the length of the Nm capsular polysaccharides isregulated by the reaction time, then the ratio of donor carbohydrate toacceptor carbohydrate is not relevant. For example, the donor:acceptorratio can be from 10:1 to 10000:1, preferably from 100:1 to 8000:1 (e.g.from 200:1 to 1000:1)

For example, if the terminally truncated CP-X is regulated by thereaction time, then the ratio of donor carbohydrate to acceptorcarbohydrate of 10:1, 20:1, 40:1, 60:1, 80:1, 100:1, 120:1, 140:1,160:1, 180:1, 200:1, 220:1, 240:1, 250:1, 300:1, 350:1, 400:1, 450:1,500:1, 550:1, 600:1, 650:1, 700:1, 750:1, 800:1, 850:1, 900:1, 950:1,1000:1, 2000:1, 3000:1, 4000:1, 5000:1, 6000:1, 7000:1, 8000:1, 9000:1,or 10000:1 may be used.

In the herein described and provided in vitro methods, if the length ofthe produced capsule polysaccharides in regulated via the reaction time,it is envisaged that the truncated version of the CP-X is used in aconcentration ranging from 20 to 500 nM, preferably from 20 to 200 nM,or more preferably from 50 to 100 nM (e.g. 50 nM). In addition, it isenvisaged herein that said truncated version of the CP-X is incubatedwith a donor concentration of 1 to 100 mM, preferably of 1 to 50 mM,more preferably of 2 to 20 mM, even more preferably of 5 to 10 mM, ormost preferably of 10 mM UDP-GlcNAc; and with an acceptor concentrationresulting in a donor to acceptor ratio from 10:1 to 10000:1, preferablyfrom 100:1 to 8000:1, (e.g. from 200:1 to 1000:1) and that the reactiontime ranges from 3 to 45 minutes, more preferably from 5 to 30 minutes,or most preferably from 5 to 10 minutes. Preferably, at least 80% of theacceptor carbohydrates are polysaccharides with a DP of ≧4.

Or, in other words, if the length of the Nm capsular polysaccharides isregulated by the reaction time, it is envisaged that the concentrationof the truncated version of the CP-X (e.g. a capsule polymerase havingat least 80% (preferably at least 90%) identity to any one of thesequences of SEQ ID NOs: 25, 28 and 29 and being functional) ranges from20 to 500 nM, preferably from 20 to 200 nM, or more preferably from 50to 100 nM (e.g. 50 nM). In addition, it is envisaged herein that saidtruncated version of the CP-X is incubated with a donor concentration of1 to 100 mM, preferably of 1 to 50 mM, more preferably of 2 to 20 mM,even more preferably of 5 to 10 mM, or most preferably of 10 mMUDP-GlcNAc; and with an acceptor concentration resulting in a donor toacceptor ratio from 10:1 to 10000:1, preferably from 100:1 to 8000:1(e.g. 200:1 to 1000:1); and that the reaction time ranges from 3 to 45minutes. Preferably, at least 80% of the acceptor carbohydrates arepolysaccharides with a DP of ≧4.

As indicated above, if the length of the Nm capsular polysaccharides isregulated by the reaction time, then the reaction time ranges (i.e. is)from 3 to 45 minutes. Preferably, the reaction time ranges from 5 to 30minutes, more preferably from 5 to 10 minutes (e.g. 5 or 10 min). Forexample, if the capsule polymerase is a polypeptide which has at least80% (preferably at least 90%) sequence identity to SEQ ID NO: 28(ΔC99-CP-X) and being functional, then the reaction time rangespreferably from 5 to 10 minutes, and is most preferably 5 minutes. Inaddition, if the capsule polymerase is a polypeptide which has at least80% (preferably at least 90%) sequence identity to SEQ ID NO: 25(ΔN58-CP-X) and being functional, then the reaction time rangespreferably from 5 to 30 minutes. Furthermore, if the capsule polymeraseis a polypeptide which has at least 80% (preferably at least 90%)sequence identity to SEQ ID NO: 29 (ΔN58ΔC99-CP-X) and being functional,then the reaction time ranges preferably from 5 to 30 minutes and ismost preferably 10 minutes.

In the inventive in vivo methods, the temperature in the incubation step(a) may be from 20° C. to 40° C., preferably from 25° C. to 37° C., morepreferably from 30° C. to 37° C., and most preferably 37° C.

As indicated above, if the length of the Nm CPS is regulated by thereaction time, the reaction time may be 3-45 min, preferably 5-30minutes. For example, the reaction time may be 3 min, 5 min, 10 min, 15min, 20 min, 25 min 30 min, 35 min, 40 min or 45 min. In addition, ifthe length of the Nm CPS is regulated by the reaction time, it ispreferred that a C- and N-terminally truncated version of the CP-X (suchas ΔN58ΔC99-CP-X) is used. This truncated version of the CP-X may be apolypeptide having the amino acid sequence of SEQ ID NO: 29 or apolypeptide having an amino acid sequence which has at least 80%homology to SEQ ID NO: 29 and being functional.

If the length of the Nm CPS is regulated by the ratio of donorcarbohydrate to acceptor carbohydrate, then the reaction time may be,e.g., 4-7 hours.

In step (b) of the herein described in vitro methods for producing NmCPS which have a defined length (i.e. which have low dispersity in termsof length), the resulting CPS are isolated. Isolation of the synthesizedCPS may be performed, e.g., by anion exchange chromatography (AEC). Forexample, the produced CPS may be purified via AEC by using a MonoQ HR5/5column (Pharmacia biotech) at a flow rate of 1 mL/min and a linearsodium chloride gradient or by High Performance Anionic ExchangeChromatography with Pulsed Amperometric Detection (HPAEC-PAD) asdescribed (Berti et al. 2012 Vaccine. (2012) 30:6409-15).

In accordance with the present invention, the ingredients which are usedin the herein described methods for producing Nm CPS can be packedtogether as a composition. Accordingly, one aspect of the inventionrelates to a composition comprising:

-   (i) at least one capsule polymerase;-   (ii) at least one donor carbohydrate; and-   (iii) at least one acceptor carbohydrate,    wherein the ratio of donor carbohydrate to acceptor carbohydrate is    a ratio from 10:1 to 10000:1 (e.g. 20:1 to 1000:1),    and wherein the capsule polymerase is the capsule polymerase of    Neisseria meningitidis serogroup A or a truncated capsule polymerase    of Neisseria meningitidis serogroup X.

The skilled person understands that, depending on the exactness of themeasurements, the exact ratio can slightly deviate from 10:1 to 10000:1.Accordingly, the methods of the invention can also be performed with aratio of about 10:1 to 10000:1. Thus, one aspect of the inventionrelates to a composition comprising:

-   (i) at least one capsule polymerase;-   (ii) at least one donor carbohydrate; and-   (iii) at least one acceptor carbohydrate,    wherein the ratio of donor carbohydrate to acceptor carbohydrate is    a ratio from about 10:1 to about 10000:1 (e.g. about 20:1 to about    1000:1),    and wherein the capsule polymerase is the capsule polymerase of    Neisseria meningitidis serogroup A or a truncated capsule polymerase    of Neisseria meningitidis serogroup X.

For example, in the herein provided compositions, 20 nM to 500 μM(preferably 20 to 200 nM, more preferably 50 to 100 nM, e.g. 50-70 nM)of the capsular polymerase (e.g. ΔC99-CP-X, ΔN58ΔC99-CP-X or ΔN58-CP-X,or a polypeptide having at least 80% (preferably at least 90%) identityto any one of SEQ ID NOs: 25, 28 and 29 and being functional) may beused. The amount of donor carbohydrate (e.g. UDP-GlcNAc) may be 1-100mM, preferably 1 to 50 mM, more preferably 2 to 20 mM (e.g. 5-10 mM).The acceptor carbohydrate may be purified capsular polysaccharides ofNmX. Preferably, the amount of the acceptor carbohydrate is adjusted toresult in a ratio of donor carbohydrate to acceptor carbohydrate of 10:1to 10000:1, preferably of 100:1 to 8000:1 (e.g. of 200:1 to 1000:1). Forexample, the amount of the acceptor carbohydrate may be 1-1000 μM, e.g.10-40 μM.

In the herein provided compositions, the ratio of donor carbohydrate toacceptor carbohydrate is 10:1 to 10000:1, e.g. 20:1, 40:1, 60:1, 80:1,100:1, 150:1, 200:1, 220:1, 240:1, 250:1, 300:1, 350:1, 400:1, 450:1,500:1, 550:1, 600:1, 650:1, 700:1, 750:1, 800:1, 850:1, 900:1, 950:1,1000:1, 2000:1, 3000:1, 4000:1, 5000:1, 6000:1, 7000:1, 8000:1, 9000:1or 10000:1. For example, the donor-acceptor-ratio may be from 2:1 to2000:1. One aspect of the invention relates to the herein describedcomposition, wherein the ratio of donor carbohydrate to acceptorcarbohydrate is a ratio from 20:1 to 240:1.

The composition of the invention may be suitable for storage (e.g.storage under cold temperatures to prevent that the synthesis ofcapsular polysaccharides starts). For instance, the composition may besuitable for storage in 4° C., 0° C., −10° C., −20° C. or −80° C.

The inventive compositions and in vitro methods comprise “at least one”capsule polymerase. Therefore, the inventive compositions and in vitromethods can comprise several capsular polymerases of different types inone reaction mixture (e.g. CP-A together with the terminally truncatedCP-X). However, it is preferred that the inventive compositions and invitro methods comprise only capsule polymerases of one type (e.g. onlyCP-A or only the terminally truncated version of CP-X).

The in vitro methods of the invention may be realized by using anappropriate kit. Accordingly, another embodiment of the inventionrelates to a kit for carrying out the in vitro methods of the invention,comprising the above described composition. The embodiments disclosed inconnection with the in vitro method of the present invention apply,mutatis mutandis, to the composition and the kit of the presentinvention.

Advantageously, the kit of the present invention further comprises,optionally (a) reaction buffer(s), storage solutions, wash solutionsand/or remaining reagents or materials required for the conduction ofthe assays as described herein. Furthermore, parts of the kit of theinvention can be packaged individually in vials or bottles or incombination in containers or multicontainer units. Thesevials/bottles/containers or multicontainers may, in addition to thecapsule polymerases and donor carbohydrates and acceptor carbohydratesas described herein, comprise preservatives or buffers for storage. Inaddition, the kit may contain instructions for use.

The composition of the present invention or the kit of the presentinvention may be advantageously used for carrying out the in vitromethods as described herein (i.e. for producing Nm capsularpolysaccharides of NmX which have a defined length). The manufacture ofthe kit of the present invention follows preferably standard procedureswhich are known to the person skilled in the art.

In the methods and compositions of the invention, the capsule polymeraseof NmA (CP-A also designated as CsaB) may be used. The DNA sequence ofthe wild type full length CP-A is shown herein as SEQ ID NO: 1. The DNAsequence of the full length CP-A which is optimized for expression in E.coli (i.e. a codon optimized DNA sequence) is shown herein as SEQ ID NO:2. The polypeptide resulting from the expression of the wild type orcodon optimized CP-A polynucleotide is identical. The amino acidsequence of the full length CP-A is given in SEQ ID NO: 9.

Of note, within the DNA sequence of the CP-A, the inventors of thepresent invention identified an additional ATG start codon and clonedthe corresponding truncation ΔN69-CP-A (which starts from thealternative ATG). The designation “ΔN69-CP-A” means that 69 amino acidsare lacking at the N-terminus of the CP-A protein. Herein, “ΔN69-CP-A”is also designated as “dN69-CsaB”. The polynucleotide sequence of acodon optimized version of ΔN69-CP-A is shown in SEQ ID NO: 3. Thepolynucleotide sequence of the wild type ΔN69-CP-A is shown in SEQ IDNO: 46. The amino acid sequence of the expressed wild type or codonoptimized ΔN69-CP-A is identical and in SEQ ID NO: 10. Surprisingly,ΔN69-CP-A is advantageous over the full length CP-A. In particular, asdemonstrated in the appended illustrative examples, while increaseddegradation and concomitantly reduced expression was seen for the codonoptimized full length CP-A, the codon optimized ΔN69-CP-A is wellexpressed and no degradation is detectable. In addition, as shown in theappended examples, by using the radioincorporation assay as described inFiebig, Glycobiology (2014) 24:150-8, it was demonstrated the ΔN69-CP-Aprotein is active (i.e. is capable of producing NmA capsularpolysaccharides). Furthermore, the appended examples also show that theΔN69-CP-A and the full length CP-A show identical activity profiles.This is surprising since several other tested truncated versions of CP-A(i.e. ΔN97-CP-A, ΔN167-CP-A, ΔN235-CP-A, ΔC45-CP-A and ΔC25-CP-A) weredemonstrated to be not active. It is mentioned that herein, inparticular in the appended examples, “A” is also designated as “d” or“delta”.

Accordingly, one aspect of the present invention relates to a truncatedversion of the CP-A (i.e. Δ69-CP-A), wherein said truncated version ofthe CP-A is the polypeptide of any one of (a) to (f):

-   -   (a) a polypeptide comprising an amino acid sequence encoded by a        nucleic acid molecule having the nucleic acid sequence of SEQ ID        NO: 3 or 46;    -   (b) a polypeptide comprising the amino acid sequence of SEQ ID        NO: 10;    -   (c) a polypeptide encoded by a nucleic acid molecule encoding a        polypeptide comprising the amino acid sequence of SEQ ID NO: 10        or of a functional fragment thereof, wherein the function        comprises the ability to transfer ManNAc-1P or a derivative        thereof from UDP-ManNAc or a derivative thereof onto an acceptor        carbohydrate;    -   (d) a polypeptide comprising an amino acid sequence encoded by a        nucleic acid molecule hybridizing under stringent conditions to        the complementary strand of a nucleic acid molecule as defined        in (a) or (c) and encoding a functional polypeptide; or a        functional fragment thereof, wherein the function comprises the        ability to transfer ManNAc-1P or a derivative thereof from        UDP-ManNAc or a derivative thereof onto an acceptor        carbohydrate;    -   (e) a polypeptide having at least 80%, preferably at least 85%,        more preferably at least 90%, even more preferably at least 95%,        even more preferably at least 96%, even more preferably at least        97%, even more preferably at least 98%, or even more preferably        at least 99% homology to the polypeptide of any one of (a) to        (d), whereby said polypeptide is functional; or a functional        fragment thereof, wherein the function comprises the ability to        transfer ManNAc-1P or a derivative thereof from UDP-ManNAc or a        derivative thereof onto an acceptor carbohydrate; and    -   (f) a polypeptide comprising an amino acid sequence encoded by a        nucleic acid molecule being degenerate as a result of the        genetic code to the nucleotide sequence of a nucleic acid        molecule as defined in (a), (c), and (d).

As indicated above, the herein provided truncated version of the CP-Acomprises the ability to transfer ManNAc-1P or a derivative thereof fromUDP-ManNAc or a derivative thereof onto an acceptor carbohydrate (i.e.onto the hydroxyl group of C6 of a capsular polysaccharide from NmA or aderivative thereof). Accordingly, this function can be the ability totransfer ManNAc-1P from UDP-ManNAc onto an acceptor carbohydrate. Theacceptor carbohydrate may be extended/activated at the reducing end orthe reducing end phosphate. In addition, the size of the producedcapsular polysaccharides can be controlled by the ratio of donor andacceptor carbohydrate when the ratio is in the range of 10:1 to 400:1,preferably from 10:1 to 240:1, more preferably from 10:1 to 80:1, evenmore preferably from 10:1 to 75:1, even more preferably from 20:1 to75:1, and most preferably from 20:1 to 60:1 (e.g. 40:1 to 60:1 such as50:1). Within this range, only one product distribution corresponding insize to the calculated donor-acceptor ratio is obtained at reactionendpoints.

The transfer of ManNAc-1P (or a derivative thereof) from UDP-ManNAc (ora derivative thereof) onto an acceptor carbohydrate may be performed byincubating a truncated version of the CP-A (i.e. ΔN69-CP-A) withUDP-ManNAc (or a derivative thereof) and an acceptor carbohydrate (e.g.a tetramer or a dimer consisting of ManNAc-1P units linked together byphosphodiester bonds). Whether a transfer from ManNAc-1P (or aderivative thereof) from UDP-ManNAc (or a derivative thereof) onto anacceptor carbohydrate has occurred can be tested, e.g. by highpercentage PAGE and developed by alcian blue/silver staining (Fiebig,Glycobiology (2014) 24:150-8). in the presence of a size marker or by³¹P NMR monitoring the characteristic phosphodiester signal, or by HighPerformance Anionic Exchange Chromatography with Pulsed AmperometricDetection (HPAEC-PAD) as described (Berti Vaccine (2012) 30:6409-15).

A derivative of ManNAc-1P may be ManNAc-3-O-Ac or ManNAc-4-O-Ac orManNAc-3,4-di-O-Ac. A derivative of UDP-ManNAc may be UDP-ManNAc-3-O-Acor UDP-ManNAc-4-O-Ac or UDP-ManNAc-3,4-di-O-Ac.

One aspect of the present invention relates to the above describedtruncated version of the CP-A (i.e. ΔN69-CP-A), wherein the functioncomprises the ability to transfer ManNAc-1P (or a derivative thereof)from UDP-ManNAc (or a derivative thereof) onto an acceptor carbohydrate,wherein the truncated version of the CP-A shows the same activityprofile as the full length CP-A. An activity profile means thatelongated products are of similar or identical dispersity.

The full length CP-A, the codon optimized CP-A, the ΔN69-CP-A or thecodon optimized ΔN69-CP-A may be used in the herein described in vitromethods for producing Nm capsular polysaccharides or compositions.Accordingly, one embodiment of the present invention relates to the invitro method of the invention or the composition of the invention,wherein the capsule polymerase of Neisseria meningitidis serogroup A isthe polypeptide of any one of (a) to (f):

-   -   (a) a polypeptide comprising an amino acid sequence encoded by a        nucleic acid molecule having the nucleic acid sequence of any        one of SEQ ID NO: 1 to 3;    -   (b) a polypeptide comprising the amino acid sequence of SEQ ID        NO: 9 or 10;    -   (c) a polypeptide encoded by a nucleic acid molecule encoding a        polypeptide comprising the amino acid sequence of SEQ ID NO: 9        or 10, or of a functional fragment thereof;    -   (d) a polypeptide comprising an amino acid sequence encoded by a        nucleic acid molecule hybridizing under stringent conditions to        the complementary strand of a nucleic acid molecule as defined        in (a) or (c) and encoding a functional polypeptide; or a        functional fragment thereof;    -   (e) a polypeptide having at least 80%, more preferably at least        85%, even more preferably at least 90%, even more preferably at        least 95%, even more preferably at least 96%, even more        preferably at least 97%, even more preferably at least 98% or        most preferably at least 99% identity to the polypeptide of any        one of (a) to (d), whereby said polypeptide is functional; or a        functional fragment thereof; and    -   (f) a polypeptide comprising an amino acid sequence encoded by a        nucleic acid molecule being degenerate as a result of the        genetic code to the nucleotide sequence of a nucleic acid        molecule as defined in (a), (c), and (d).

A particular embodiment of the present invention relates to the in vitromethod of the invention or the composition of the invention, wherein thecapsule polymerase of Neisseria meningitidis serogroup A is thepolypeptide of any one of (a) to (f):

-   -   (a) a polypeptide comprising an amino acid sequence encoded by a        nucleic acid molecule having the nucleic acid sequence of any        one of SEQ ID NO: 1 to 3;    -   (b) a polypeptide comprising the amino acid sequence of SEQ ID        NO: 9 or 10;    -   (c) a polypeptide encoded by a nucleic acid molecule encoding a        polypeptide comprising the amino acid sequence of SEQ ID NO: 9        or 10, or of a functional fragment thereof, wherein the function        comprises the ability to transfer ManNAc-1P or a derivative        thereof from UDP-ManNAc or a derivative thereof onto an acceptor        carbohydrate;    -   (d) a polypeptide comprising an amino acid sequence encoded by a        nucleic acid molecule hybridizing under stringent conditions to        the complementary strand of a nucleic acid molecule as defined        in (a) or (c) and encoding a functional polypeptide; or a        functional fragment thereof, wherein the function comprises the        ability to transfer ManNAc-1P or a derivative thereof from        UDP-ManNAc or a derivative thereof onto an acceptor        carbohydrate;    -   (e) a polypeptide having at least 80%, more preferably at least        85%, even more preferably at least 90%, even more preferably at        least 95%, even more preferably at least 96%, even more        preferably at least 97%, even more preferably at least 98% or        most preferably at least 99% identity to the polypeptide of any        one of (a) to (d), whereby said polypeptide is functional; or a        functional fragment thereof, wherein the function comprises the        ability to transfer ManNAc-1P or a derivative thereof from        UDP-ManNAc or a derivative thereof onto an acceptor        carbohydrate; and    -   (f) a polypeptide comprising an amino acid sequence encoded by a        nucleic acid molecule being degenerate as a result of the        genetic code to the nucleotide sequence of a nucleic acid        molecule as defined in (a), (c), and (d).

As indicated, the function of the above under items (a) to (f) describedcapsule polymerase of Neisseria meningitidis serogroup A comprises theability to transfer ManNAc-1P or a derivative thereof from UDP-ManNAc ora derivative thereof onto an acceptor carbohydrate. Preferably, at least80% of the acceptor carbohydrates are polysaccharides with a DP of ≧4.For example, the acceptor (or at least 80% of the acceptorcarbohydrates) may be DP4, DP5 or DP6; preferably the acceptor (or atleast 80% of the acceptor carbohydrates) is a tetramer or pentamer ofManNAc-1P with a free reducing end.

As described, the full length CP-A, the codon optimized CP-A or thecodon optimized ΔN69-CP-A (i.e. the polypeptide described above underitems (a) to (f)) may be used in the herein described in vitro methodsfor producing Nm capsular polysaccharides or in the herein describedcompositions. For example, if one of these capsule polymerases is usedin the in vitro methods or compositions described herein, than the ratioof donor carbohydrate to acceptor carbohydrate is from 10:1 to 400:1,preferably from 10:1 to 240:1, and more preferably from 10:1 to 80:1,and even more preferably from 10:1 to 75:1, even more preferably from20:1 to 75:1, and most preferably from 20:1 to 60:1 (e.g. 40:1 to 60:1such as 50:1).

In a prioritized aspect of the invention, the codon optimized ΔN69-CP-Ais used in the herein described in vitro methods for producing Nm CPSwhich have a defined length or in the herein described compositions.

The inventors of the present invention surprisingly identified truncatedversions of the capsule polymerase of Nm serogroup X (CP-X). Forexample, the CP-X-fragment ΔN67ΔC99-CP-X (wherein 67 amino acids arelacking at the N-terminus and 99 amino acids are lacking at theC-terminus) was demonstrated to be functional (i.e. able to transferGlcNAc-1P (or a derivative thereof) from UDP-GlcNAc (or a derivativethereof) onto an acceptor carbohydrate. Preferably, at least 80% of theacceptor carbohydrates are polysaccharides with a DP of ≧4. For example,the acceptor (or at least 80% of the acceptor carbohydrates) may be DP4,DP5, or DP6 (see, e.g., FIG. 3E); preferably, the acceptor (or at least80% of the acceptor carbohydrates) is a tetramer or pentamer ofGlcNAc-1P with a free reducing end as shown in FIG. 3. The DNA sequenceof ΔN67ΔC99-CP-X is provided herein as SEQ ID NO: 23 and the amino acidsequence is shown herein as SEQ ID NO: 31. In context of the presentinvention it is expected that the C-terminally truncated ΔC136-CP-X(wherein 136 amino acids are lacking at the C-terminus) is active, asseveral C-terminal mutations (except of a mutation at position 351) donot affect activity of the CP-X (unpublished data, see also FIG. 6C). Inaddition also the fragment ΔN83ΔC136-CP-X (wherein 83 amino acids arelacking at the N-terminus and 136 amino acids are lacking at theC-terminus) is considered to be active because the CP-A is still activeif it carries a mutation at amino acid 250 (in the N-terminus) whichcorresponds to amino acid 84 in CP-X.

As demonstrated in the appended examples, the inventors of the presentinvention surprisingly found that, when terminally truncated, the CP-Xcan be regulated via the donor-acceptor ratio such that it produces CPSwith a uniform and defined length. Furthermore, these terminallytruncated versions of the CP-X have the additional advantage that theycan also be regulated by the reaction time. Moreover, the inventiveterminally truncated versions of CP-X have the superior property thatthey have a significantly improved activity as compared to the fulllength CP-X. The DNA sequence of the full length CP-X is shown herein asSEQ ID NO: 16 and the amino acid sequence of the full length CP-X isshown herein as SEQ ID NO: 24.

The illustrative appended examples demonstrate several truncatedversions of the CP-X which are capable of producing NmX capsularpolysaccharides. For example, the proteins ΔN58-CP-X (wherein 58 aminoacids are lacking at the N-terminus of CP-X and which comprisesapproximately 88% of the full length CP-X), ΔC99-CP-X (wherein 98 aminoacids are lacking at the C-terminus of CP-X and which comprisesapproximately 80% of the full length CP-X) and ΔN58ΔC99-CP-X (wherein 58amino acids are lacking at the N-terminus and 98 amino acids are lackingat the C-terminus and which comprises approximately 68% of the fulllength CP-X) have been demonstrated to be functional capsularpolysaccharides. Herein, in particular in the appended examples,“ΔN58-CP-X”, “ΔC99-CP-X” and “ΔN58ΔC99-CP-X” are also designated as“dN58-CsxA”, “dC99-CsxA” and “dN58dC99-CsxA”, respectively. Regardingthe designation “ΔC99” it is noted that this designation has been chosenas the DNA sequence which was used to generate the ΔC99-CP-X constructends with codon 387 of the CP-X sequence. However, this DNA sequence wascloned to have an XhoI restriction site (CTCGAG) at the 3′ end. Thecodon CTC within this site encodes leucine. Amino acid 388 of wild typeCP-X is also a leucine (however encoded by CTT). Therefore, in theproteins ΔC99-CP-X and ΔN58ΔC99-CP-X, 98 (instead of 99) amino acids arelacking at the C-terminus. In this regard it is further noted that ifthe herein provided polynucleotide encoding ΔC99-CP-X (i.e. SEQ ID NO:20) is expressed in a host cell, then the resulting polypeptide has thesequence as given herein in SEQ ID NO: 28. Analogously, if the hereinprovided polynucleotide encoding ΔN58ΔC99-CP-X (i.e. SEQ ID NO: 21) isexpressed in a host cell, then the resulting polypeptide has thesequence as given herein in SEQ ID NO: 29. Herein, in particular in theappended examples, “ΔC99-CP-X” and “ΔN58ΔC99-CP-X” are also designatedas “dC99-CsxA” and “dN58dC99-CsxA”, respectively.

As mentioned, the inventors of the present invention surprisingly foundthat truncated versions of the CP-X have a considerably higher activityas compared to the full length CP-X. More specifically, by using aradioincorporation assay, the appended illustrative examples demonstratethat truncated versions of the CP-X (e.g. ΔN58-CP-X, ΔC99-CP-X orΔN58ΔC99-CP-X) are more active (i.e. assemble more saccharide monomersto capsule polysaccharides) as compared to the full length CP-X. In theherein described and provided in vitro methods and compositions, thetruncated version of the CP-X may preferably be a terminally truncatedversion of the CP-X. Accordingly, a further embodiment of the presentinvention relates to a truncated version of the CP-X, which is apolypeptide comprising:

-   -   (a) an amino acid sequence encoded by a nucleic acid molecule        which is a terminally truncated version of the nucleic acid        sequence of SEQ ID NO: 16 and comprises maximal 50-95% of the        nucleic acid sequence of SEQ ID NO: 16;    -   (b) an amino acid sequence which is a terminally truncated        version of the amino acid sequence of SEQ ID NO: 24 and        comprises maximal 50-95% of the amino acid sequence of SEQ ID        NO: 24;    -   (c) a polypeptide encoded by a nucleic acid molecule encoding a        polypeptide having an amino acid sequence which is a terminally        truncated version of the amino acid sequence of SEQ ID NO: 24        and comprises maximal 50-95% of the amino acid sequence of SEQ        ID NO: 24;    -   (d) a polypeptide encoded by a nucleic acid molecule hybridizing        under stringent conditions to the complementary strand of a        nucleic acid molecule as defined in (a) or (c) and encoding a        functional polypeptide;    -   (e) a polypeptide having at least 80%, more preferably at least        85%, even more preferably at least 90%, even more preferably at        least 95%, even more preferably at least 96%, even more        preferably at least 97%, even more preferably at least 98% or        most preferably at least 99% identity to the polypeptide of any        one of (a) to (d), whereby said polypeptide is functional; or    -   (f) a polypeptide comprising an amino acid sequence encoded by a        nucleic acid molecule being degenerate as a result of the        genetic code to the nucleotide sequence of a nucleic acid        molecule as defined in (a), (c) or (d).

The function of the terminally truncated version of the CP-X comprisesthe ability to transfer GlcNAc-1P from UDP-GlcNAc onto an acceptorcarbohydrate. Preferably, at least 80% of the acceptor carbohydrates arepolysaccharides with a DP of ≧4. For example, the acceptor (or at least80% of the acceptor carbohydrates) may be DP4, DP5, or DP6; preferably,the acceptor (or at least 80% of the acceptor carbohydrates) is atetramer or pentamer of GlcNAc-1P with a free reducing end as shown inFIG. 3. The terminally truncated version of CP-X which is applied hereinhas also the ability to transfer derivatives of GlcNAc-1P fromderivatives of UDP-GlcNAc onto an acceptor carbohydrate. Thus, thefunction of the terminally truncated version of the CP-X comprises theability to transfer GlcNAc-1P or a derivative thereof from UDP-GlcNAc ora derivative thereof onto an acceptor carbohydrate. Therefore, thepresent invention also relates to a truncated version of the CP-X, whichis a polypeptide comprising:

-   -   (a) an amino acid sequence encoded by a nucleic acid molecule        which is a terminally truncated version of the nucleic acid        sequence of SEQ ID NO: 16 and comprises maximal 50-95% of the        nucleic acid sequence of SEQ ID NO: 16;    -   (b) an amino acid sequence which is a terminally truncated        version of the amino acid sequence of SEQ ID NO: 24 and        comprises maximal 50-95% of the amino acid sequence of SEQ ID        NO: 24;    -   (c) a polypeptide encoded by a nucleic acid molecule encoding a        polypeptide having an amino acid sequence which is a terminally        truncated version of the amino acid sequence of SEQ ID NO: 24        and comprises maximal 50-95% of the amino acid sequence of SEQ        ID NO: 24;    -   (d) a polypeptide encoded by a nucleic acid molecule hybridizing        under stringent conditions to the complementary strand of a        nucleic acid molecule as defined in (a) or (c) and encoding a        functional polypeptide; wherein the function comprises the        ability to transfer GlcNAc-1P or a derivative thereof from        UDP-GlcNAc or a derivative thereof onto an acceptor        carbohydrate;    -   (e) a polypeptide having at least 80%, more preferably at least        85%, even more preferably at least 90%, even more preferably at        least 95%, even more preferably at least 96%, even more        preferably at least 97%, even more preferably at least 98% or        most preferably at least 99% identity to the polypeptide of any        one of (a) to (d), whereby said polypeptide is functional;        wherein the function comprises the ability to transfer GlcNAc-1P        or a derivative thereof from UDP-GlcNAc or a derivative thereof        onto an acceptor carbohydrate; or    -   (f) a polypeptide comprising an amino acid sequence encoded by a        nucleic acid molecule being degenerate as a result of the        genetic code to the nucleotide sequence of a nucleic acid        molecule as defined in (a), (c) or (d).

As described above, the herein provided terminally truncated version ofthe CP-X comprises maximal 50-95% of the full length CP-X. It isenvisaged, that the terminally truncated version of the CP-X comprisesminimal 50% of the full length CP-X and maximal 95% of the full lengthCP-X. For example, the terminally truncated CP-X of the invention maycomprise 50-95%, 55-90%, 60-85% or 65-80% of the full length CP-X. Forexample, the terminally truncated CP-X may comprise 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90% or 95% or the full length CP-X.

The herein provided terminally truncated version of the CP-X comprisesthe ability to transfer GlcNAc-1P (or a derivative thereof) fromUDP-GlcNAc (or a derivative thereof) onto an acceptor carbohydrate (i.e.onto the hydroxyl group of C4 of a capsular polysaccharide from NmX (ora derivative thereof)). The acceptor carbohydrate may beextended/activated at the reducing end or the reducing end phosphate.Preferably, at least 80% of the acceptor carbohydrates arepolysaccharides with a DP of ≧4. For example, the acceptor (or at least80% of the acceptor carbohydrates) may be DP4, DP5, or DP6; preferably,the acceptor (or at least 80% of the acceptor carbohydrates) is atetramer or pentamer of GlcNAc-1P with a free reducing end as shown inFIG. 3. In addition, the size of the produced capsular polysaccharidescan be controlled by the ratio of donor and acceptor carbohydrate whenthe ratio is in the range of 10:1-400:1. Within this range, only oneproduct distribution with a low dispersity (compared to full-lengthCP-X) is obtained at reaction endpoints. More specifically, within thisrange, only one product distribution with a low dispersity is obtainedduring all time points of the reaction.

The transfer GlcNAc-1P (or a derivative thereof) from UDP-GlcNAc (or aderivative thereof) onto an acceptor carbohydrate may be performed byincubating a truncated version of the CP-X (e.g. ΔC99-CP-X, ΔN58-CP-X orΔN58ΔC99-CP-X) with UDP-GlcNAc (or a derivative thereof) and an acceptorcarbohydrate and in reaction buffer (50 mM Tris pH 8.0, and 20 mM MgCl₂,and optionally 1-2 mM (e.g. 1 mM) DTT) and incubated for 1 h—over night(preferably for 4-7 hours) at 25-37° C. (preferably between 30 and 37°C.) (Fiebig et al., 2014; Glycobiology. 2014 February; 24(2):150-8).Preferably, at least 80% of the acceptor carbohydrates arepolysaccharides with a DP of ≧4. For example, the acceptor (or at least80% of the acceptor carbohydrates) may be DP4, DP5, or DP6; preferably,the acceptor (or at least 80% of the acceptor carbohydrates) is atetramer or pentamer of GlcNAc-1P with a free reducing end as shown inFIG. 3.

Whether a transfer from GlcNAc-1P (or a derivative thereof) fromUDP-GlcNAc (or a derivative thereof) onto an acceptor carbohydrate hasoccurred can be tested, e.g. by high percentage PAGE and developed byalcian blue/silver staining (Fiebig et al., Glycobiology. 2014 February;24(2):150-8) in the presence of a size marker or by ³¹P NMR monitoringthe characteristic phosphodiester signal (Fiebig et al., Glycobiology.2014 February; 24(2):150-8), or by High Performance Anionic ExchangeChromatography with Pulsed Amperometric Detection (HPAEC-PAD) asdescribed (Berti et al. Vaccine. 2012 Oct. 5; 30(45):6409-15; Fiebig etal., Glycobiology. 2014 February; 24(2):150-8).

In context of the present invention it was surprisingly found that theC-terminally truncated CP-X as well as the C- and N-terminally truncatedCP-X can be regulated via the donor-acceptor ratio and/or via thereaction time so as to produce capsular polysaccharides that have auniform and defined length. In particular, the appended examples showthat the truncated proteins ΔC99-CP-X and ΔN58ΔC99-CP-X can be regulatedvia the donor-acceptor ratio or via the reaction time such that theyproduce capsular polysaccharides which have a uniform and definedlength. Accordingly, one aspect the invention relates to a terminallytruncated version of the CP-X, which is a C-terminally or a C- andN-terminally truncated version of the full length CP-X.

Thus, the invention relates to the herein provided terminally truncatedversion of the CP-X, comprising:

-   -   (a) an amino acid sequence which is a C-terminally or C- and        N-terminally truncated version of the amino acid sequence of SEQ        ID NO: 24 and comprises maximal 50-95% of the amino acid        sequence of SEQ ID NO: 24;    -   (b) a polypeptide encoded by a nucleic acid molecule encoding a        polypeptide having an amino acid sequence which is a        C-terminally or C- and N-terminally truncated version of the        amino acid sequence of SEQ ID NO: 24 and comprises maximal        50-95% of the amino acid sequence of SEQ ID NO: 24; or    -   (c) a polypeptide having at least 80%, more preferably at least        85%, even more preferably at least 90%, even more preferably at        least 95%, even more preferably at least 96%, even more        preferably at least 97%, even more preferably at least 98% or        most preferably at least 99% identity to the polypeptide of (a)        or (b), whereby said polypeptide is functional; wherein the        function comprises the ability to transfer GlcNAc-1P or a        derivative thereof from UDP-GlcNAc or a derivative thereof onto        an acceptor carbohydrate.

In one aspect of the present invention, the terminally truncated versionof the CP-X which is described and provided herein is the constructΔN58-CP-X, ΔC99-CP-X or ΔN58ΔC99-CP-X.

Thus, the invention relates to the herein provided terminally truncatedversion of the CP-X, comprising

-   -   (a) a polypeptide comprising an amino acid sequence encoded by a        nucleic acid molecule having the nucleic acid sequence of any        one of SEQ ID NO: 17, 20 or 21;    -   (b) a polypeptide comprising the amino acid sequence of any one        of SEQ ID NO: 25, 28 or 29;    -   (c) a polypeptide comprising an amino acid sequence encoded by a        nucleic acid molecule hybridizing under stringent conditions to        the complementary strand of a nucleic acid molecule as defined        in (a) and encoding a functional polypeptide; wherein the        function comprises the ability to transfer GlcNAc-1P or a        derivative thereof from UDP-GlcNAc or a derivative thereof onto        an acceptor carbohydrate;    -   (d) a polypeptide having at least 80%, more preferably at least        85%, even more preferably at least 90%, even more preferably at        least 95%, even more preferably at least 96%, even more        preferably at least 97%, even more preferably at least 98% or        most preferably at least 99% identity to the polypeptide of any        one of (a) to (c), whereby said polypeptide is functional;        wherein the function comprises the ability to transfer GlcNAc-1P        or a derivative thereof from UDP-GlcNAc or a derivative thereof        onto an acceptor carbohydrate; and    -   (e) a polypeptide comprising an amino acid sequence encoded by a        nucleic acid molecule being degenerate as a result of the        genetic code to the nucleotide sequence of a nucleic acid        molecule as defined in (a) or (c).

Accordingly, in one aspect the invention relates to the terminallytruncated version of the CP-X comprising:

-   -   (i) an amino acid sequence encoded by a nucleic acid molecule        which comprises the nucleic acid sequence of SEQ ID NO: 17, 20        or 21; or a nucleic acid sequence having at least 80%, more        preferably at least 85%, even more preferably at least 90%, even        more preferably at least 95%, even more preferably at least 96%,        even more preferably at least 97%, even more preferably at least        98% or most preferably at least 99% identity to the nucleic acid        sequence of any one of SEQ ID NO: 17, 20 or 21 and encoding a        functional polypeptide; wherein the function comprises the        ability to transfer GlcNAc-1P or a derivative thereof from        UDP-GlcNAc or a derivative thereof onto an acceptor        carbohydrate; or    -   (ii) the amino acid sequence of SEQ ID NO: 25, 28 or 29; or an        amino acid sequence having at least 80%, more preferably at        least 85%, even more preferably at least 90%, even more        preferably at least 95%, even more preferably at least 96%, even        more preferably at least 97%, even more preferably at least 98%        or most preferably at least 99% identity to any one of SEQ ID        NOs: 25, 28 or 29 and being functional, wherein the function        comprises the ability to transfer GlcNAc-1P or a derivative        thereof from UDP-GlcNAc or a derivative thereof onto an acceptor        carbohydrate.

A particular embodiment of the invention relates to the above describedterminally truncated version of the CP-X, wherein the function comprisesthe ability to transfer GlcNAc-1P or a derivative thereof fromUDP-GlcNAc or a derivative thereof onto an acceptor carbohydrate;wherein the produced polymers have a DP between 10 and 60 or an avDPbetween 15 and 20, when the ratio of donor carbohydrate to acceptorcarbohydrate is in the range of 10:1 to 400:1. Preferably, at least 80%of the acceptor carbohydrates are polysaccharides with a DP of ≧4. Forexample, the acceptor (or at least 80% of the acceptor carbohydrates)may be DP4, DP5, or DP6; preferably, the acceptor (or at least 80% ofthe acceptor carbohydrates) is a tetramer or pentamer of GlcNAc-1P witha free reducing end as shown in FIG. 3.

As described, the terminally truncated version of the CP-X (i.e. thepolypeptide described above under items (i) and (ii)) may be used in theherein described in vitro methods for producing Nm capsularpolysaccharides or in the herein described compositions.

For example, if the terminally truncated CP-X is regulated via the ratioof donor carbohydrate to acceptor carbohydrate (and not via the reactiontime) then the following preferred ratios can produce a productdistribution with a low dispersity. Or, in other words, if theterminally truncated version of the CP-X is regulated via the ratio ofdonor carbohydrate to acceptor carbohydrate, then the followingpreferred ratios can produce capsular polysaccharides of which at least60%, (preferably at least 80%) have a defined length between DP10 andDP60 or between avDP15 and avDP20. Moreover, the following capsulepolymerases and ratios are preferably used in the compositions describedherein.

For example, if the capsule polymerase is a polypeptide which has atleast 80% (preferably at least 85%, even more preferably at least 90%,even more preferably at least 95%, even more preferably at least 96%,even more preferably at least 97%, even more preferably at least 98% ormost preferably at least 99%) sequence identity to a polypeptide encodedby a nucleic acid molecule which comprises the nucleic acid sequence ofSEQ ID NO: 20 (ΔC99-CP-X) and being functional, then the ratio of donorcarbohydrate to acceptor carbohydrate is preferably from 10:1 to 80:1,more preferably from 20:1 to 60:1, even more preferably from 20:1 to50:1, and most preferably from 33:1 to 50:1. In addition, if the capsulepolymerase is a polypeptide which has at least 80% (preferably at least85%, even more preferably at least 90%, even more preferably at least95%, even more preferably at least 96%, even more preferably at least97%, even more preferably at least 98% or most preferably at least 99%)sequence identity to a polypeptide encoded by a nucleic acid moleculewhich comprises the nucleic acid sequence of SEQ ID NO: 17 (ΔN58-CP-X)and being functional, then the ratio of donor carbohydrate to acceptorcarbohydrate is preferably from 10:1 to 80:1, more preferably from 20:1to 50:1, and most preferably from 20:1 to 33:1 (e.g. 33:1). Furthermore,if the capsule polymerase is a polypeptide which has at least 80%(preferably at least 85%, even more preferably at least 90%, even morepreferably at least 95%, even more preferably at least 96%, even morepreferably at least 97%, even more preferably at least 98% or mostpreferably at least 99%) sequence identity to a polypeptide encoded by anucleic acid molecule which comprises the nucleic acid sequence of SEQID NO: 21 (ΔN58ΔC99-CP-X) and being functional, then the ratio of donorcarbohydrate to acceptor carbohydrate is preferably from 50:1 to 400:1,more preferably from 80:1 to 400:1, even more preferably from 100:1 to400:1, and most preferably from 100:1 to 200:1. In addition, if thecapsule polymerase is a polypeptide which comprises an amino acidsequence which has at least 80% (preferably at least 85%, even morepreferably at least 90%, even more preferably at least 95%, even morepreferably at least 96%, even more preferably at least 97%, even morepreferably at least 98% or most preferably at least 99%) sequenceidentity to SEQ ID NO: 28 (ΔC99-CP-X) and being functional, then theratio of donor carbohydrate to acceptor carbohydrate is preferably from10:1 to 80:1, more preferably from 20:1 to 60:1, even more preferablyfrom 20:1 to 50:1, and most preferably from 33:1 to 50:1. Furthermore,if the capsule polymerase is a polypeptide which comprises an amino acidsequence which has at least 80% (preferably at least 85%, even morepreferably at least 90%, even more preferably at least 95%, even morepreferably at least 96%, even more preferably at least 97%, even morepreferably at least 98% or most preferably at least 99%) sequenceidentity to SEQ ID NO: 25 (ΔN58-CP-X) and being functional, then theratio of donor carbohydrate to acceptor carbohydrate is preferably from10:1 to 80:1, more preferably from 20:1 to 50:1, and most preferablyfrom 20:1 to 33:1 (e.g. 33:1). Moreover, if the capsule polymerase is apolypeptide which comprises an amino acid sequence which has at least80% (preferably at least 85%, even more preferably at least 90%, evenmore preferably at least 95%, even more preferably at least 96%, evenmore preferably at least 97%, even more preferably at least 98% or mostpreferably at least 99%) sequence identity to SEQ ID NO: 29(ΔN58ΔC99-CP-X) and being functional, then the ratio of donorcarbohydrate to acceptor carbohydrate is preferably from 50:1 to 400:1,more preferably from 80:1 to 400:1, even more preferably from 100:1 to400:1, and most preferably from 100:1 to 200:1.

In context of the invention the terminally truncated version of the CP-Xmay also be a polypeptide comprising the construct ΔN65ΔC10-CP-X (whichis shown in SEQ ID NOs: 22 and 30) or ΔN67ΔC99-CP-X (which is shown inSEQ ID NOs: 23 and 31) which have been demonstrated to be active (datanot shown) and which can be regulated via the donor-acceptor ratio orvia the reaction time such that they produce capsular polysaccharideswith a uniform and defined length.

Accordingly, the terminally truncated version of the CP-X of the presentinvention may be a polypeptide comprising the amino acid of any one ofSEQ ID NOs: 25, 28, 29, 30 or 31. Also encompassed by the presentinvention are terminally truncated versions of the CP-X comprising theamino acid sequence of any one of SEQ ID NOs: 25, 28, 29, 30 or 31wherein one, two, three or more amino acid residues are added, deletedor substituted. The polypeptides may have the function of the terminallytruncated version of the CP-X (i.e. the ability to transfer GlcNAc-1P ora derivative thereof from UDP-GlcNAc or a derivative thereof onto anacceptor carbohydrate). Preferably, at least 80% of the acceptorcarbohydrates are polysaccharides with a DP of ≧4. The amino acidsequence of the polypeptides may be at least 80%, more preferably atleast 85%, more preferably at least 90%, more preferably at least 95%,more preferably at least 96%, more preferably at least 97%, morepreferably at least 98%, and most preferably at least 99% identical toSEQ ID NO: 25, 28, 29, 30 or 31. The polypeptides may have the functionof a truncated version of the CP-X. Preferably, the function comprisesthe ability to transfer GlcNAc-1P (or a derivative thereof) fromUDP-GlcNAc (or a derivative thereof) onto an acceptor carbohydrate;wherein at least 60% (preferably at least 80%) of the produced polymershave a DP between 10 and 60 or an avDP between 15 and 20, when the ratioof donor carbohydrate to acceptor carbohydrate is in the range of 10:1to 400:1. Preferably, at least 80% of the acceptor carbohydrates arepolysaccharides with a DP of ≧4. For example, the acceptor (or at least80% of the acceptor carbohydrates) may be DP4, DP5, or DP6; preferably,the acceptor (or at least 80% of the acceptor carbohydrates) is atetramer or pentamer of GlcNAc-1P with a free reducing end as shown inFIG. 3.

The herein described terminally truncated versions of the CP-X may belinked (e.g. operatively linked) to additional, heterologous sequences.E.g. the nucleic acid molecule encoding a truncated version of the CP-Xmay be linked to additional, heterologous nucleic acid sequences in arecombinant nucleic acid molecule. For example, said additional nucleicacid sequence may be a coding gene.

The term “recombinant nucleic acid molecule” relates to nucleic acidmolecules originating from a different genetic context and combined bymolecular biological methods. Here, the term “different genetic context”relates to genomes from different species, varieties or individuals ordifferent positions within a genome. Recombinant nucleic acid moleculescan contain not only natural sequences but also sequences, which,compared to the natural ones are mutated or chemically modified or else,the sequences are altogether newly synthesized sequences.

As indicated above, the terminally truncated version of the CP-X may belinked to an additional heterologous molecule. Said additionalheterologous molecule may be a nucleic acid molecule which is preferablyoperatively linked to a truncated version of the CP-X. Said additionalheterologous nucleic acid molecule may originate from a differentgenetic context than the truncated version of the CP-X. Non-limitingexamples of additional heterologous molecules comprise in particularmarker molecules, like luciferase, galactosidase, GFP, EGFP, DsRed, etc.or tag-molecules, like Flag-tags, CBP and others. In a particular aspectof the invention, said additional heterologous molecule is a tag, suchas a StrepII-tag, a thrombin-tag, a his-tag, a MBP-tag or a S3N10-tag.In a further aspect of the invention, said additional heterologousmolecule is a nucleic acid molecule encoding a restriction site (e.g. arestriction site of the enzymes BamHI/BglII, XhoI or NdeI). It is alsoenvisaged that the truncated CP-X of the invention is linked to severaladditional sequences (e.g. several tags or several restriction sites).Yet, as detailed below, also sequences which optimize (e.g. enhance) theexpression of the terminally truncated CP-X may be operatively linked tothe nucleic acid sequence of the truncated CP-X. Such a sequence may bea particular promoter sequence. A suitable promoter for the expressionof the terminally truncated version of the CP-X may be, e.g., Tac-, T7-,or Lacpromoter.

One embodiment of the present invention relates to a nucleic acidmolecule encoding the herein described truncated version of the CP-X.The invention also relates to a vector comprising said nucleic acidmolecule. The present invention further relates to vectors containing anucleic acid molecule of the present invention encoding a terminallytruncated version of the CP-X. The present invention relates also to avector comprising the nucleic acid construct encoding the hereindescribed truncated version of the CP-X.

The term “vector” relates to circular or linear nucleic acid moleculeswhich can autonomously replicate in host cells into which they areintroduced. The “vector” as used herein particularly refers to plasmids,cosmids, viruses, bacteriophages and other vectors commonly used ingenetic engineering. In a preferred embodiment, the vectors of theinvention are suitable for the transformation of cells, like fungalcells, cells of microorganisms such as yeast or prokaryotic cells. In aparticularly preferred embodiment such vectors are suitable for stabletransformation of bacterial cells, for example to express the truncatedversion of the CP-X of the present invention.

Accordingly, in one aspect of the invention, the vector as provided isan expression vector. Generally, expression vectors have been widelydescribed in the literature. As a rule, they may not only contain aselection marker gene and a replication-origin ensuring replication inthe host selected, but also a promoter, and in most cases a terminationsignal for transcription. Between the promoter and the terminationsignal there is preferably at least one restriction site or a polylinkerwhich enables the insertion of a nucleic acid sequence/molecule desiredto be expressed.

It is to be understood that when the vector provided herein is generatedby taking advantage of an expression vector known in the prior art thatalready comprises a promoter suitable to be employed in context of thisinvention (for example expression of a truncated version of the CP-X asdescribed herein above) the nucleic acid construct is inserted into thatvector in a manner that the resulting vector comprises only one promotersuitable to be employed in context of this invention. The skilled personknows how such insertion can be put into practice. For example, thepromoter can be excised either from the nucleic acid construct or fromthe expression vector prior to ligation.

A non-limiting example of the vector of the present invention is theplasmid vector pET22b comprising a nucleic acid construct of the presentinvention. Further examples of vectors suitable to comprise a nucleicacid construct of the present invention to form the vector of thepresent invention are known in the art and are, for example othervectors for bacterial expression systems such as vectors of the pETseries (Novagen) or pQE vectors (Qiagen).

In an additional embodiment, the present invention relates to a hostcell comprising the herein described vector. In particular, theinvention relates to a host cell comprising a nucleic acid constructencoding a terminally truncated version of the CP-X and/or the vector ofthe present invention. Preferably, the host cell of the presentinvention may be a prokaryotic cell, for example, a bacterial cell. As anon limiting example, the host cell of the present invention may beEscherichia coli. The host cell provided herein is intended to beparticularly useful for generating the terminally truncated version ofthe CP-X of the present invention.

Generally, the host cell of the present invention may be a prokaryoticor eukaryotic cell, comprising a nucleic acid construct of the inventionor the vector of the invention or a cell derived from such a cell andcontaining a nucleic acid construct of the invention or the vector ofthe invention. In a preferred embodiment, the host cell is geneticallymodified with a nucleic acid construct of the invention or the vector ofthe invention in such a way that it contains the nucleic acid constructof the present invention integrated into the genome. For example, suchhost cell of the invention, but also the host cell of the invention ingeneral, may be a bacterial, yeast, or fungus cell.

In one particular aspect, the host cell of the present invention iscapable to express or expresses a terminally truncated version of theCP-X as defined herein and as illustrative characterized in SEQ ID NOs:17, 20, 21, 22 or 23, for example in SEQ ID NO: 21. An overview ofexamples of different corresponding expression systems to be used forgenerating the host cell of the present invention is for instancecontained in Methods in Enzymology 153 (1987), 385-516, in Bitter et al.(Methods in Enzymology 153 (1987), 516-544), in Sawers et al. (AppliedMicrobiology and Biotechnology 46 (1996), 1-9), Billman-Jacobe (CurrentOpinion in Biotechnology 7 (1996), 500-4), Hockney (Trends inBiotechnology 12 (1994), 456-463), and in Griffiths et al., (Methods inMolecular Biology 75 (1997), 427-440).

The transformation or genetically engineering of the host cell with anucleic acid construct of the invention or the vector according to theinvention can be carried out by standard methods, as for instancedescribed in Sambrook and Russell (2001), Molecular Cloning: ALaboratory Manual, CSH Press, Cold Spring Harbor, N.Y., USA; Methods inYeast Genetics, A Laboratory Course Manual, Cold Spring HarborLaboratory Press, 1990.

The nucleic acid molecules, vectors, host cells and polypeptidesdescribed herein may be used for producing (i.e. synthesizing) Nmcapsular polysaccharides which have a defined length.

As mentioned, in the herein provided in vitro methods for producing NmCPS which have a defined length or in the compositions of the invention,the terminally truncated version of the CP-X as described herein may beused.

As for vaccine production the Nm CPS have preferably a DP of 10 to 60(i.e. an avDP of 15 to 20), it is preferred that the Nm CPS which areproduced by the herein described in vitro methods have this size.Advantageously, if in the herein described in vitro methods the CP-A ora terminally truncated version of the CP-X is used, then the produced NmCPS have a DP or 10 to 60 (i.e. an avDP of 15 to 20), if thedonor-to-acceptor-ratio is in the range from 10:1 to 400:1. In addition,if in herein described in vitro methods the truncated version of theCP-X is used, then the produced Nm CPS have a DP of 10 to 60 (i.e. anavDP of 15 to 20) if the incubation time ranges from 3 to 45 minutes.Thus, one aspect of the invention relates to the herein provided invitro methods, wherein the capsular polysaccharides have a DP of 10 to60 or an avDP of 15 to 20. In a preferred aspect of the herein providedin vitro methods, at least 60% (more preferably at least 80%) of thecapsular polysaccharides have a DP of 10 to 60 or an avDP of 15 to 20.In addition, a further embodiment of the invention relates to the invitro methods of the invention or the composition of the invention,wherein

-   -   (i) the ratio of donor carbohydrate to acceptor carbohydrate is        a ratio from 20:1 to 80:1; and the capsule polymerase is either        a polypeptide comprising maximal 80% of the amino acid sequence        of SEQ ID NO: 24 (CP-X) and comprising the amino acid sequence        of SEQ ID NO: 28 (ΔC99-CP-X), or a polypeptide comprising the        amino acid sequence of SEQ ID NO: 9 (CP-A); or    -   (ii) the ratio of donor carbohydrate to acceptor carbohydrate is        a ratio from 100:1 to 400:1; and the capsule polymerase is a        polypeptide comprising maximal 68% of the amino acid sequence of        SEQ ID NO: 24 (CP-X) and comprising the amino acid sequence of        SEQ ID NO: 29 (ΔN58ΔC99-CP-X).

In item (i) of the above described embodiment, the ratio of donorcarbohydrate to acceptor carbohydrate may be 50:1.

According to the inventive in vitro method, the donor carbohydrate whichis contacted with at least one purified capsule polymerase (CP) mayfurther be activated during step (a). Thus, one embodiment of theinvention relates to the in vitro methods of the invention, wherein saiddonor carbohydrate is activated or wherein said donor carbohydrate isactivated during step (a). Said activation during step (a) may beperformed by contacting said donor carbohydrate with an activatingenzyme. In addition, during this in step (a) of the inventive in vitromethods, said donor carbohydrate may be further contacted with PEPand/or at least one activating nucleotide. Analogously, in the hereinprovided composition said donor carbohydrate may be activated. In themethods or in the composition of the invention, said donor carbohydratemay be activated by linkage to an activating nucleotide. Said activatingnucleotide may be, e.g. CMP, CDP, CTP, UMP, UDP, TDP, AMP or UTP,preferably UDP.

For example, the activation may be mediated by linkage of an activatingnucleotide such as CMP, UDP, TDP or AMP. Preferably, the activatingnucleotide is UDP. The activation of a carbohydrate by linkage of anucleotide may be catalyzed by several activating enzymes which areknown in the art. Such activating enzymes may be incubated with the atleast one donor carbohydrate, at least one acceptor carbohydrate and theat least one CP during step (a) of the in vitro method provided herein.UDP-ManNAc is preferably synthesized from UDP-GlcNAc using the enzymeUDP-GlcNAc-epimerase. In SEQ ID NO: 32, the nucleotide sequence ofUDP-GlcNAc-epimerase cloned from Neisseria meningitidis serogroup A isshown, the corresponding polypeptide sequence of UDP-GlcNAc-epimerase isshown in SEQ ID NO: 33.

One particular embodiment of the present invention relates to the hereinprovided in vitro methods or the herein provided composition, whereinthe capsule polymerase is a truncated version of the capsule polymeraseof Neisseria meningitidis serogroup X and wherein at least one donorcarbohydrate is UDP-GlcNAc. However, another embodiment of the presentinvention relates to the herein provided in vitro methods or the hereinprovided composition, wherein the capsule polymerase is a truncatedversion of the capsule polymerase of Neisseria meningitidis serogroup Xand wherein at least one donor carbohydrate is GlcNAc-1-P.

In a further embodiment of the herein described in vitro methods orcompositions the capsule polymerase is the capsule polymerase ofNeisseria meningitidis serogroup A and at least one donor carbohydrateis UDP-ManNAc. The sugar building block UDP-ManNAc is commercially notavailable. Therefore, a further advantage of the present invention isthat UDP-ManNAc can be synthesized (during the inventive in vitro methodfor producing Nm capsular polysaccharides) from cheap UDP-GlcNAc. Thus,another embodiment of the present invention relates to the inventive invitro methods or the inventive composition, wherein the capsulepolymerase is the capsule polymerase of Neisseria meningitidis serogroupA and wherein at least one donor carbohydrate is UDP-GlcNAc.

In context of the present invention, UDP-ManNAc is preferablysynthesized from UDP-GlcNAc using the enzyme UDP-GlcNAc-epimerase. InSEQ ID NO: 32, the nucleotide sequence of UDP-GlcNAc-epimerase clonedfrom Neisseria meningitidis serogroup A is shown, the correspondingpolypeptide sequence of UDP-GlcNAc-epimerase is shown in SEQ ID NO: 33.Thus, one aspect of the invention relates to the herein provided invitro methods, wherein in step (a) the capsule polymerase is furtherincubated with the UDP-GlcNAc-epimerase. In addition, one embodiment ofthe invention relates to the herein provided composition, furthercomprising the UDP-GlcNAc-epimerase. For example, in the in vitromethods or compositions of the present invention, theUDP-GlcNAc-epimerase (e.g. CsaA of NmA) may be used in a concentrationof 1-2000 nmol, e.g. 10 nmol.

In another embodiment of the inventive in vitro methods andcompositions, the capsular polymerase (CP) may be a truncated version ofthe CP-X or a functional derivative thereof and at least one donorcarbohydrate may be GlcNAc-1-phosphate. Said donor carbohydrateGlcNAc-1-phosphate may be further contacted with at least one nucleotideand/or phosphoenolpyruvate (PEP) and auxiliary enzymes. Said nucleotidecan be, e.g., UMP, UDP or UTP. Said donor carbohydrateGlcNAc-1-phosphate may further be activated during incubation with theterminally truncated CP-X. In accordance with the herein presented invitro method, this activation may yield the activated sugar nucleotideUDP-GlcNAc.

Thus, the CP to be applied in the means and methods described herein maybe a truncated version of CP-X or a functional derivative thereof and atleast one donor carbohydrate may be UDP-GlcNAc or a derivative thereof.Examples for derivatives of UDP-GlcNAc may be compounds that arealkylated or hydroxylated or that comprise additional functional groups,such as carboxylic acids, azides, amides, acetyl groups or halogenatoms; see also “Carbohydrate chemistry” Volumes 1-34, Cambridge[England], Royal Society of Chemistry, loc. cit.

Generally, in context of the present invention, the saccharidesdescribed herein may also be labelled forms of these saccharides. Forexample, the saccharides may be labelled radioactively, such as [¹⁴C] or[³H]. Such labelling may be inter alia useful in diagnostic applicationsand uses of the saccharides described herein. Such diagnosticapplications and uses will be further described herein below.

In accordance with the inventive method, the terminally truncatedversion of the CP-X is contacted with at least one donor carbohydrateand with at least one acceptor carbohydrate during the incubation step(a) of the in vitro method presented herein. Said acceptor carbohydratemay be oligomeric or polymeric CPS of Neisseria meningitidis serogroup X(CPSX), and/or a carbohydrate structure containing terminal GlcNAcresidues such as hyaluronic acid, heparin, heparin sulphate orprotein-linked oligosaccharides.

In the herein provided methods for producing Nm capsular polysaccharideswhich have a defined length, it is preferred that the acceptorcarbohydrate has a DP≧4. Or, in other words, acceptor carbohydrates witha length of DP≧4 are particularly preferred in the herein describedmethods for producing Nm capsular polysaccharides which have a definedlength. Normally, the acceptor carbohydrates are purified capsularpolysaccharides of the respective serogroup and have different length.However, in the herein provided methods for producing capsularpolysaccharides with a defined length, and in the herein providedcompositions, it is preferred that at least 80% of the acceptorcarbohydrates are polysaccharides with a DP of ≧4. The acceptorcarbohydrates may also consist of polysaccharides with a DP of ≧4. Forexample, if the CP-A is used, then the acceptor (or at least 80% of theacceptor carbohydrates) is preferably DP4, DP5, or DP6; more preferablythe acceptor (or at least 80% of the acceptor carbohydrates) is atetramer or pentamer of ManNAc-1P with a free reducing end. If aterminally truncated version of the CP-X is used, then the acceptor (orat least 80% of the acceptor carbohydrates) is preferably DP4, DP5, orDP6; more preferably, the acceptor (or at least 80% of the acceptorcarbohydrates) is a tetramer or pentamer of GlcNAc-1P with a freereducing end as shown in FIG. 3.

An example of the present invention relates to the in vitro method forproducing Nm capsular polysaccharides of NmX which have a definedlength, said method comprising the steps:

-   (a) incubating a truncated version of the CP-X with UDP-GlcNAc and    hydrolysed capsular polysaccharides of NmX; wherein the ratio of    UDP-GlcNAc to hydrolysed capsular polysaccharides of NmX is a ratio    from 10:1 to 400:1 (e.g. 20:1 to 400:1); and-   (b) isolating the resulting capsular polysaccharide.

A further example of the present invention relates to the in vitromethod for producing Nm capsular polysaccharides of NmX which have adefined length, said method comprising the steps:

-   (a) incubating a truncated version of the CP-X with UDP-GlcNAc and    hydrolysed capsular polysaccharides of NmX; wherein the incubation    time ranges from 3 to 45 minutes; and-   (b) isolating the resulting capsular polysaccharide.

Another example of the present invention relates to the in vitro methodfor producing Nm capsular polysaccharides of NmX which have a definedlength, said method comprising the steps:

-   (a) incubating a truncated version of the CP-X with UDP-GlcNAc and    hydrolysed capsular polysaccharides of NmX; wherein    -   (i) the ratio of donor carbohydrate to acceptor carbohydrate is        a ratio from 10:1 to 10000 (e.g. 200:1 to 1000:1), and    -   (ii) the incubation time ranges from 3 to 45 minutes; and-   (b) isolating the resulting capsular polysaccharide.

In the above examples also other combinations of activated ornon-activated donor carbohydrates and acceptor carbohydrates asdescribed herein can be applied. Such other combinations do not deferfrom the gist of the present invention.

In one embodiment of the present in vitro method, the CP to be used isCP-A or a functional derivative thereof and at least one donorcarbohydrate may be UDP-ManNAc or a derivative thereof. Examples forderivatives of UDP-ManNAc may be compounds that are alkylated orhydroxylated or that comprise additional functional groups such ascarboxylic acids, azides, amides, acetyl groups or halogen atoms; seealso “Carbohydrate chemistry” Volumes 1-34: monosaccharides,disaccharides, and specific oligosaccharides, Reviews of the literaturepublished during 1967-2000, Cambridge (England), Royal Society ofChemistry.

In another embodiment of the in vitro method described herein, the CP isCP-A or a functional derivative thereof and at least one donorcarbohydrate is ManNAc-1-phosphate or a derivative thereof. Examples forderivatives of ManNAc-1-phosphate ManNAc-3-O-Ac or ManNAc-4-O-Ac orManNAc-3,4-di-O-Ac. In the herein provided in vitro method, said donorcarbohydrate ManNAc-1-phosphate (ManNAc-1-P) may be contacted with atleast one nucleotide and/or phosphoenolpyruvate (PEP) and auxiliaryenzymes during step (a) of the in vitro method. Said nucleotide can be,e.g., UMP, UDP and UTP. Said donor carbohydrate ManNAc-1-phosphate maybe activated during incubation with CP-A. In accordance with the hereinpresented in vitro method, this activation may yield the activated sugarnucleotide UDP-ManNAc, or its derivatives UDP-ManNAc-3-O-Ac orUDP-ManNAc-4-O-Ac or UDP-ManNAc-3,4-di-O-Ac. Thus, one aspect of theinvention relates to the herein provided in vitro methods, wherein thecapsule polymerase is the capsule polymerase of Neisseria meningitidisserogroup A and wherein at least one donor carbohydrate is ManNAc-1-P.

In the herein provided in vitro method, CP-A or a functional derivativethereof is contacted with at least one donor carbohydrate and with anacceptor carbohydrate during the incubation step (a) of the inventive invitro method. In accordance with the inventive in vitro method presentedherein, the acceptor carbohydrate may be oligomeric or polymeric CPS ofNeisseria meningitidis serogroup A (CPSA) and/or a carbohydratestructure containing terminal GlcNAc or ManNAc residues such ashyaluronic acid, heparin, heparin sulphate or protein-linkedoligosaccharides. As described above, in the herein provided in vitromethods, the acceptor carbohydrate of CP-A may be the cheap and easilycommercially available UDP-GlcNAc, as the UDP-GlcNAc-epimerase can beused to convert UDP-GlcNAc into UDP-ManNAc.

An example of the present invention relates to the in vitro method forproducing Nm capsular polysaccharides of NmA which have a definedlength, said method comprising the steps:

-   (a) incubating CP-A with UDP-GlcNAc, UDP-GlcNAc-epimerase and    hydrolysed capsular polysaccharides of NmA; wherein the ratio of    UDP-GlcNAc to hydrolysed capsular polysaccharides of NmA is a ratio    from 10:1 to 400:1 (e.g. 20:1 to 400:1); and-   (b) isolating the resulting capsular polysaccharide.

A further example of the present invention relates to the in vitromethod for producing Nm capsular polysaccharides of NmA which have adefined length, said method comprising the steps:

-   (a) incubating CP-A with UDP-ManNAc and hydrolysed capsular    polysaccharides of NmA; wherein the ratio of UDP-ManNAc to    hydrolysed capsular polysaccharides of NmA is a ratio from 10:1 to    400:1 (e.g. 20:1 to 400:1); and-   (b) isolating the resulting capsular polysaccharide.

Again, the skilled person readily understands that also othercombinations of activated or non-activated donor carbohydrates andacceptor carbohydrates as described herein can be applied. Such othercombinations do not defer from the gist of the present invention.

As described herein and illustrated in the appended examples, theacceptor carbohydrate is preferred by the CP (e.g. CP-A) if it ispresented in non-acetylated form. Moreover, the acceptor is even morepreferred if presented in non-acetylated, de-phosphorylated form.Accordingly, one embodiment of the present invention relates to theherein provided in vitro methods or the herein provided composition,wherein the acceptor carbohydrate is non-acetylated and/ordephosphorylated, preferably non-acetylated and dephosphorylated.

Within a CPS obtainable by the inventive method described hereinabove,one or more carbohydrates of the CPS-subunits may be derivatized and maycontain, for example, additional functional groups such as acetylgroups, amino groups, alkyl groups, hydroxyl groups, carboxylic acids,azides, amides, or halogen atoms; see also “Carbohydrate chemistry”Volumes 1-34: monosaccharides, disaccharides, and specificoligosaccharides, Reviews of the literature published during 1967-2000,Cambridge (England), Royal Society of Chemistry. Thus, in one embodimentof the inventive in vitro method or composition, the acceptorcarbohydrate carries one or more additional functional groups at itsreducing end. Said additional functional groups, may be, e.g., acetylgroups, carboxylic acids, azides, amides, or halogen atoms; see also“Carbohydrate chemistry” Volumes 1-34, Cambridge [England], RoyalSociety of Chemistry, loc. cit.

One embodiment of the present invention relates to the herein providedin vitro method or the herein provided composition, wherein saidacceptor carbohydrate is capsule polysaccharide of Neisseriameningitidis serogroup A or X or a carbohydrate structure containingterminal GlcNAc residues. Said carbohydrate structure containingterminal GlcNAc-residues may be hyaluronic acid, heparin sulphate,heparan sulphate or protein-linked oligosaccharides.

As demonstrated in the appended examples, the inventors of the presentinvention analyzed the minimal length of the priming acceptor for CP-A.Therefore, synthetic compounds which are shown in FIG. 25 which vary notonly in length, but also with respect to O-acetylation (compounds 2, 4,6 are 3-O-acetylated) and reducing end modifications have been used. Incompounds 1, 2, 5, 6 the reducing ends are occupied by adecyl-phosphate-ester, while a methyl group (OMe) is present in thecompounds 3 and 4. As evident from the appended examples, the activityof the codon optimized ΔN69-CP-A did not go beyond background (noacceptor) with compounds 1-4, but, intriguingly, steeply increased withdisaccharides carrying a decyl-phosphate-ester at the reducing end(compounds 5, 6) (FIG. 25). With the non-acetylated compound 5, activityvalues similar to those obtained with the optimized acceptorCPSA_(hyd(deOAc))-deP were measured. Interestingly, O-acetylation ofcompound 5 (resulting in compound 6) reduced the quality of theacceptor. Based on these data, the minimal acceptor recognized by CP-Aor a derivative of CP-A (such as ΔN69-CP-A) can be defined as the dimerof ManNAc-1P units linked together by phosphodiester bonds. The presenceof a phosphodiester at the reducing end seems obligatory, becausecompounds ending with OMe groups are not used by the capsule polymerase.

Thus, in context of the present invention, a dimer of ManNAc-1P carryinga phosphodiester at the reducing end was identified as minimal acceptorfor CP-A or derivatives of CP-A (such as ΔN69-CP-A).

Accordingly, the inventors of the present invention identified twoimportant features of CP-A: (i) the minimal efficient acceptor is adimer and (ii) the reducing end phosphate group can be extended withrather large chemical groups (such as decyl-ester). This latter findingbears the perspective that chain elongation can be primed with reagentsof very high purity and functional groups that facilitate conjugation ofglycans to carrier proteins in the vaccine production chain (Costantino(2011) Expert opinion on drug discovery 6 (10), 1045-1066; Bardotti(2008) Vaccine 26 (18), 2284-2296). Besides decyl-ester, chainelongation may be primed with a number of functional groups that allowconjugation to carrier proteins, e.g., Alkyl-azides, Alkyl-amindes orsulfhydryl-reagents chemical functions as described in Costantino etal., 2011 Expt. Opin. Drug. Discov. 6:1053; Table 2.

Accordingly, one embodiment of the present invention relates to the invitro method of the invention or the composition of the invention,wherein said acceptor carbohydrate is a dimer of ManNAc carrying aphosphodiester at the reducing end. In this embodiment of the presentinvention, the capsule polymerase may be the capsule polymerase ofNeisseria meningitidis serogroup A. Moreover, the phosphate group at thereducing end may be extended with alkyl-azides, alkyl-amindes orsulfhydryl-groups and chemical functions as described in Costantino(2011) Expt. Opin. Drug. Discov. 6:1053; Table 2. For example, in theherein provided in vitro methods or composition, the acceptorcarbohydrate may be a disaccharide carrying a decyl-phosphate-ester atthe reducing end. As mentioned, in this embodiment of the presentinvention, the capsule polymerase may be the capsule polymerase ofNeisseria meningitidis serogroup A.

If in the herein described in vitro methods or compositions the acceptoris a dimer of ManNAc carrying a phosphodiester at the reducing end(which may be extended with large chemical groups such as adecyl-ester), then the capsular polymerase may be CP-A or a derivativeof CP-A (such as ΔN69-CP-A).

The capsule polymerase (CP) which is incubated with the at least onedonor carbohydrate in the presented in vitro methods and compositionsmay be purified. Said CP (or a functional fragment or derivativethereof) may be isolated from Neisseria meningitidis lysates orrecombinantly produced. Recombinant production of a CP (or a functionalfragment or derivative thereof) may be performed by transferring the DNAcoding for the CP (or the functional fragment or derivative thereof)into a vector and using a host (e.g. eukaryotic host cells such asinsect cells or yeast or prokaryotic host cells such as Escherichiacoli) to express the CP (or the functional fragment or derivativethereof). After expression, the CP (or the functional fragment orderivative thereof) may be purified from the cell lysate. Thepurification may be carried out following the instructions of theQiaexpressionist (http://www.qiagen.com/literature/render.aspx?id=128,Qiagen) for bacterial protein purification or by using BD BioscienceBaculo Gold instruction (Cat. No. 560138) for insect cell proteinproduction and purification. For instance, the CP (or the functionalfragment or derivative thereof) may be produced by purifying therecombinant CP (or the functional fragment or derivative thereof) frombacteria via his-tag purification following the instructions of theQiaexpressionist. A CP (or a functional fragment or derivative thereof)may also be synthetically or chemically produced. Therefore solid phasepeptide synthesis (SPPS) may be applied.

The donor carbohydrates which are used in the herein provided means andmethods may be obtained from commercial sources or may be chemicallysynthesized (Wolf, Chemistry (2009) 15:7656-64; Wolf, Eur J Cell Biol.(2010) 89:63-75. The acceptor carbohydrates which are used in theinventive means and methods may be obtained by purifying them fromNeisseria meningitidis cultures and can be chemically synthesized asdescribed in the attached manuscript (Black (2010) Synthesis ofstructures corresponding to the capsular polysaccharide of Neisseriameningitidis group A. 4th Baltic Meeting on Microbial Carbohydrates,Abstracts, p. 56 (abstract 5), Hyytiälä Forestry Field Station, Finland;Black (2011) Towards synthetic glycoconjugates based on the structure ofNeisseria meningitidis group A capsular polysaccharide. 16th EuropeanCarbohydrate Symposium, Abstracts, p. 61 (OL-02), Sorrento, Italy;Nikolaev (2011) From phosphosaccharide chemistry to potentialanti-parasite and anti-bacterial carbohydrate vaccines. CarbohydrateGordon Research Conference, Abstracts, p. 4, Colby College, Waterville,Me., USA.). One embodiment of the invention relates to the hereinprovided in vitro methods or composition, wherein said acceptorcarbohydrate is purified.

As mentioned, the acceptor carbohydrate which is contacted with thedonor carbohydrate and the CP may be purified according to the in vitromethod described herein. If said acceptor carbohydrate is oligomeric orpolymeric CPS of Neisseria meningitidis, it may be hydrolysed. Thus, oneaspect of the invention relates to the herein provided in vitro methodsor composition, wherein said acceptor capsule polysaccharide ishydrolysed.

It is known in the art that capsule polysaccharides of NmA areimmunogenic only if O-acetylated (Berry (2002) Infection and immunity 70(7), 3707-3713). Therefore, in the herein described in vitro methods forproducing capsular polysaccharides an O-acetyltransferase can be usedwhich is able to perform this modification in a nature identical form.Accordingly, one embodiment of the invention relates to the hereinprovided in vitro method, further comprising O-acetylation of theproduced capsule polysaccharides. Said O-acetylation may be performed bycontacting the produced capsule polysaccharides with anO-acetyltransferase. Also provided herein is the composition of theinvention, further comprising an O-acetyltransferase.

The O-acetyltransferase which is used in the inventive methods andcompositions may be, e.g. the CsaC of NmA. Accordingly, one aspect ofthe invention relates to the in vitro methods of the invention or thecomposition of the invention, wherein, wherein the O-acetyltransferaseis the polypeptide of any one of (a) to (f):

-   -   (a) a polypeptide comprising an amino acid sequence encoded by a        nucleic acid molecule having the nucleic acid sequence of SEQ ID        NO: 53;    -   (b) a polypeptide comprising the amino acid sequence of SEQ ID        NO: 54;    -   (c) a polypeptide encoded by a nucleic acid molecule encoding a        polypeptide comprising the amino acid sequence of SEQ ID NO: 54        or of a functional fragment thereof;    -   (d) a polypeptide comprising an amino acid sequence encoded by a        nucleic acid molecule hybridizing under stringent conditions to        the complementary strand of a nucleic acid molecule as defined        in (a) or (c) and encoding a functional polypeptide; or a        functional fragment thereof;    -   (e) a polypeptide having at least 80%, more preferably at least        85%, even more preferably at least 90%, even more preferably at        least 95%, even more preferably at least 96%, even more        preferably at least 97%, even more preferably at least 98% or        most preferably at least 99% identity to the polypeptide of any        one of (a) to (d), whereby said polypeptide is functional; or a        functional fragment thereof; and    -   (f) a polypeptide comprising an amino acid sequence encoded by a        nucleic acid molecule being degenerate as a result of the        genetic code to the nucleotide sequence of a nucleic acid        molecule as defined in (a), (c), and (d).

The function of this O-acetyltransferase comprises the ability totransfer Acetyl-groups from the donor Acetyl-Coenzyme A ontohydroxyl-groups of UDP-ManNAc or oligo- and polymeric structuresconsisting of ManNAc-1-phosphate units linked together by phosphodiesterlinkages. Or, in other words this O-acetlytransferase catalyses theformation of an ester-bond between an Acetyl-group and a hydroxyl-groupof ManNAc-1-phosphate containing molecule. Thus, one aspect of theinvention relates to the in vitro methods of the invention or thecomposition of the invention, wherein the O-acetyltransferase is thepolypeptide of any one of (a) to (f):

-   -   (a) a polypeptide comprising an amino acid sequence encoded by a        nucleic acid molecule having the nucleic acid sequence of SEQ ID        NO: 53;    -   (b) a polypeptide comprising the amino acid sequence of SEQ ID        NO: 54;    -   (c) a polypeptide encoded by a nucleic acid molecule encoding a        polypeptide comprising the amino acid sequence of SEQ ID NO: 54        or of a functional fragment thereof, wherein the function        comprises the ability to transfer Acetyl-groups from the donor        Acetyl-Coenzyme A onto hydroxyl-groups of UDP-ManNAc or oligo-        and polymeric structures consisting of ManNAc-1-phosphate units        linked together by phosphodiester linkages;    -   (d) a polypeptide comprising an amino acid sequence encoded by a        nucleic acid molecule hybridizing under stringent conditions to        the complementary strand of a nucleic acid molecule as defined        in (a) or (c) and encoding a functional polypeptide; or a        functional fragment thereof, wherein the function comprises the        ability to transfer Acetyl-groups from the donor Acetyl-Coenzyme        A onto hydroxyl-groups of UDP-ManNAc or oligo- and polymeric        structures consisting of ManNAc-1-phosphate units linked        together by phosphodiester linkages;    -   (e) a polypeptide having at least 80%, more preferably at least        85%, even more preferably at least 90%, even more preferably at        least 95%, even more preferably at least 96%, even more        preferably at least 97%, even more preferably at least 98% or        most preferably at least 99% identity to the polypeptide of any        one of (a) to (d), whereby said polypeptide is functional; or a        functional fragment thereof, wherein the function comprises the        ability to transfer Acetyl-groups from the donor Acetyl-Coenzyme        A onto hydroxyl-groups of UDP-ManNAc or oligo- and polymeric        structures consisting of ManNAc-1-phosphate units linked        together by phosphodiester linkages; and    -   (f) a polypeptide comprising an amino acid sequence encoded by a        nucleic acid molecule being degenerate as a result of the        genetic code to the nucleotide sequence of a nucleic acid        molecule as defined in (a), (c), and (d).

In context of the present invention, production and O-acetylation ofcapsular polysaccharides (e.g. of capsular polysaccharides of NmA) maybe performed by using a two-step protocol or in a one-pot reaction.

For example, the O-acetylation of capsular polysaccharides may beperformed by incubating in vitro synthesized capsular polysaccharides(e.g. 1 mg) in the presence of an O-acetyltransferase (e.g. 1.2 nmolCsaC of NmA) in a total volume of 0.5 mL. The reaction may be performedin reaction buffer (e.g. a buffer comprising 25 mM Tris pH 7.5 and 50 mMNaCl), started by the addition of an equimolar amount of Acetyl-CoA(e.g. 14 mM final) and allowed to proceed for 4 h at 37° C. Obviously,this protocol may be up or down scale depending on the needed amount ofO-acetylated capsular polysaccharides.

As mentioned above, capsule polysaccharides of NmA are only immunogenicif they are O-acetylated. In addition, natural capsule polysaccharidesof NmA are O-acetylated in positions 3 and 4 of ManNAc. Thus, if theherein provided in vitro methods are combined with O-acetylation of theproduced capsule polysaccharides, then the used capsule polymerase maybe CP-A. Thus, one aspect of the invention relates to the hereinprovided in vitro methods or composition, wherein the capsule polymeraseis the capsule polymerase of Neisseria meningitidis serogroup A. Thecapsule polysaccharides produced by the inventive in vitro methods maybe O-acetylated in positions 3 and 4 of ManNAc.

As described herein, the capsule polysaccharides (e.g. the O-acetylatedcapsular polysaccharides) which are produced by the herein describedmethods may be used for glycoconjugate vaccine production. Thus, oneembodiment of the present invention relates to a method for producing avaccine comprising the herein provided in vitro method. Said method forproducing a vaccine may further comprise covalently attaching theproduced capsular polysaccharides to a carrier molecule. Said carriermolecule may be tetanus toxoid or CRM₁₉₇ (Cross-Reacting Material 197),an inactive form of diphteria-toxin (Micoli et al., 2013; PNAS).

Thus, the invention relates to a method for the production of a vaccineagainst a strain genus Neisseria comprising the steps of:

-   (a) In vitro production of (a) Nm capsule polysaccharide(s) as    defined above; and-   (b) combining said Nm capsule polysaccharide(s) with a    pharmaceutically acceptable carrier.

Accordingly, the invention relates to a method for the production of avaccine against a strain or strains of the genus Neisseria, inparticular N. meningitidis by combining (a) Nm capsularpolysaccharide(s) of the invention with a biologically acceptablecarrier.

Another embodiment of the present invention also relates to a capsularpolysaccharide which has been produced by the in vitro method of thepresent invention. The CPS obtainable by the in vitro methods describedhereinabove are useful as pharmaceuticals, e.g., as vaccines. Inparticular, the herein described CPS are advantageous as vaccines in theprophylaxis and treatment of diseases caused by Neisseria meningitidis,such as neisserial meningitidis. Accordingly, one embodiment of thepresent invention relates to the capsular polysaccharide of theinvention for use as a medicament.

As mentioned, the synthetic CPS obtainable by the inventive in vitromethod may be used as vaccines. In a preferred embodiment of the presentinvention, they are used in vaccination of a human subject. Alsodisclosed is the use of the CPS obtainable by the inventive in vitromethod for the preparation of a vaccine. In a specific embodiment of thepresent invention, the CPS obtainable by the in vitro methods describedherein are used as vaccines against meningococcal meningitidis caused byNeisseria meningitidis serogroup A or X. Thus, the medicaments disclosedherein may be useful in pharmaceutical and vaccination purposes, i.e.for the treatment of Neisseria-induced diseases or the vaccinationagainst these pathogens. Therefore, one embodiment of the presentinvention relates to a vaccine comprising the capsular polysaccharide ofthe invention, optionally further comprising a pharmaceuticallyacceptable carrier. Also provided herein is the capsular polysaccharideof the invention or the vaccine of the invention for use in vaccinationof a subject. Accordingly, the present invention provides for a methodfor treating and/or preventing a meningococcal meningitidis comprisingthe administration of (a) capsular polysaccharide(s) of the presentinvention or the vaccine(s) of the present invention to a subject inneed of such a treatment. Preferably said subject is a human patient.

A “patient” or “subject” for the purposes of the present inventionincludes both humans and other animals, particularly mammals, and otherorganisms. Thus, the methods are applicable to both human therapy andveterinary applications. In the preferred embodiment the patient is amammal, and in the most preferred embodiment the patient is human being.As indicated, the herein provided capsular polysaccharide(s) orvaccine(s) may be used for the vaccination against meningococcalmeningitidis caused by Neisseria meningitidis serogroup A or X.

The medicaments provided herein are pharmaceutical compositions and maycomprise the CPS of the present invention. The pharmacologicalcompositions may further comprise antibodies specifically directedagainst the CPS of the present invention. Such CPS as well as theantibodies directed against the same may be used, inter alia, invaccination protocols, either alone or in combination. Therefore, thepharmaceutical compositions of the present invention which comprise theCPS of the invention or antibodies directed against these CPS, may beused for pharmaceutical purposes such as effective therapy of infectedhumans or animals and/or, preferably for vaccination purposes.Accordingly, the present invention relates to pharmaceuticalcompositions comprising the CPS as described herein and/or antibodies orantibody fragments against the CPS as described herein and, optionally,a pharmaceutically acceptable carrier. In context with the presentinvention, the pharmaceutical compositions described herein may be used,inter alia, for the treatment and/or prevention of Neisseria-induceddiseases and/or infections. Preferably, the pharmaceutical compositionof the invention is used as a vaccine as will be further describedherein below.

As mentioned, the present invention also relates to pharmaceuticalcompositions comprising the capsular polysaccharides described herein.Said capsular polysaccharides may be isolated but it is also envisagedthat these capsular polysaccharides are to be used in context with otherstructures, e.g., toxins, adjuvants and the like. Such toxins may, interalia, function as carriers for the capsular polysaccharides produced bythe herein described methods. Numerous methods have been developed tolink oligosaccharides covalently to carriers (Lit: (a) Vince Pozsgay,Oligosaccharide-protein conjugates as vaccine candidates againstbacteria, Advances in Carbohydrate Chemistry and Biochemistry, AcademicPress, 2000, Volume 56, Pages 153-199, (b) Jennings, H. J., R. K. Sood(1994) Synthetic glycoconjugates as human vaccines; in Lee, Y. C. R. T.Lee (eds): Neoglycoconjugates. Preparation and Applications. San Diego,Academic Press, pp 325-371, (c) Pozsgay, V.; Kubler-Kielb, J.,Conjugation Methods toward Synthetic Vaccines, Carbohydrate-BasedVaccines, American Chemical Society, Jul. 2, 2008, 36-70); (D) Carl E.Frasch, Preparation of bacterial polysaccharide-protein conjugates:Analytical and manufacturing challenges, Vaccine, In Press, CorrectedProof, Available online 24 Jun. 2009, ISSN 0264-410X, DOI:10.1016/j.vaccine.2009.06.013.) One example is the covalent coupling ofthe synthetic CPS molecules provided herein to protein amino-groups bymeans of reductive amination. As indicated above, for producing avaccine, the herein produced CPS may be coupled to tetanus toxoid orother carrier proteins to generate a conjugate vaccine.

Thus, the capsular polysaccharide produced by the herein describedmethods may preferably used as a vaccine, i.e. the compounds providedherein can be employed for the vaccination of a subject. Such a subjectmay be a mammal and, in a particular embodiment, a human being. Thevaccines provided herein are particularly useful in the vaccinationagainst Neisseria.

The medicament/pharmaceutical composition (e.g. the vaccine) of thepresent invention comprises the CPS produced by the in vitro methods ofthe invention and may further comprise a pharmaceutically acceptablecarrier, excipient and/or diluent. Examples of suitable pharmaceuticalcarriers are well known in the art and include phosphate buffered salinesolutions, water, emulsions, such as oil/water emulsions, various typesof wetting agents, sterile solutions etc. Compositions comprising suchcarriers can be formulated by well known conventional methods. Thesepharmaceutical compositions can be administered to the subject at asuitable dose. Administration of the suitable compositions may beeffected by different ways, e.g., by intravenous, intraperitoneal,subcutaneous, intramuscular, topical, intradermal, intranasal orintrabronchial administration. The dosage regimen will be determined bythe attending physician and clinical factors. As is well known in themedical arts, dosages for any one patient depends upon many factors,including the patient's size, body surface area, age, the particularcompound to be administered, sex, time and route of administration,general health, and other drugs being administered concurrently. Thepharmaceutical composition of the present invention, particularly whenused for vaccination purposes, may be employed at about 0.01 μg to 1 gCPS per dose, or about 0.5 μg to 500 μg CPS per dose, or about 1 μg to300 μg CPS per dose. However, doses below or above this exemplary rangesare envisioned, especially considering the aforementioned factors.Administration of the suitable compositions may be effected by differentways, e.g., by intravenous, intraperitoneal, subcutaneous,intramuscular, topical or intradermal administration. However, inparticular in the pharmaceutical intervention of the present invention,Neisseria infections can demand an administration to the side ofinfection, like the brain. Progress can be monitored by periodicassessment. The compositions of the invention may be administeredlocally or systemically. Administration will generally be parenterally,e.g., intravenously. The compositions of the invention may also beadministered directly to the target site, e.g., by biolistic delivery toan internal or external target site or by catheter to a site in anartery. Preparations for parenteral administration include sterileaqueous or non-aqueous solutions, suspensions, and emulsions. Examplesof non-aqueous solvents are propylene glycol, polyethylene glycol,vegetable oils such as olive oil, and injectable organic esters such asethyl oleate. Aqueous carriers include water, alcoholic/aqueoussolutions, emulsions or suspensions, including saline and bufferedmedia. Parenteral vehicles include sodium chloride solution, Ringer'sdextrose, dextrose and sodium chloride, lactated Ringer's, or fixedoils. Intravenous vehicles include fluid and nutrient replenishers,electrolyte replenishers (such as those based on Ringer's dextrose), andthe like. Preservatives and other additives may also be present such as,for example, antimicrobials, anti-oxidants, chelating agents, and inertgases and the like. Furthermore, the pharmaceutical composition of theinvention may comprise further agents such as interleukins and/orinterferons depending on the intended use of the pharmaceuticalcomposition. The pharmaceutical composition (e.g. the vaccine) of theinvention may also be administered as co-therapy together with at leastone other active agent. This at least other active agent may be amedicament which is conventionally used as adjuvant or for preventingand/or treating Nm infections. The other active agent may be, e.g., avaccine, an antibiotic, an anti-inflammatory agent, an interleukin or aninterferon.

In a preferred embodiment of the present invention, the pharmaceuticalcomposition as defined herein is a vaccine.

Vaccines may be prepared, inter alia, from one or more CPS as describedherein, or from one or more antibodies as described herein, i.e.antibodies against the CPS as disclosed herein. Accordingly, in contextwith the present invention, vaccines may comprise one or more CPS asdescribed herein and/or one or more antibodies, fragments of saidantibodies or derivatives of the antibodies of the invention, i.e.antibodies against the CPS as disclosed herein.

The CPS or the antibodies, fragments or derivatives of said antibodiesof the invention which are used in a pharmaceutical composition may beformulated, e.g., as neutral or salt forms. Pharmaceutically acceptablesalts, such as acid addition salts, and others, are known in the art.Vaccines can be, inter alia, used for the treatment and/or theprevention of an infection with pathogens, e.g. Neisseria, and areadministered in dosages compatible with the method of formulation, andin such amounts that will be pharmacologically effective forprophylactic or therapeutic treatments.

A vaccination protocol can comprise active or passive immunization,whereby active immunization entails the administration of an antigen orantigens (like the capsule polysaccharides of the present invention orantibodies directed against these CPS) to the subject/patient in anattempt to elicit a protective immune response. Passive immunizationentails the transfer of preformed immunoglobulins or derivatives orfragments thereof (e.g., the antibodies, the derivatives or fragmentsthereof of the present invention, i.e. specific antibodies directedagainst the CPS obtained by the means and methods provided herein) to asubject/patient. Principles and practice of vaccination and vaccines areknown to the skilled artisan, see, for example, in Paul, “FundamentalImmunology” Raven Press, New York (1989) or Morein, “Concepts in VaccineDevelopment”, ed: S. H. E. Kaufmann, Walter de Gruyter, Berlin, N.Y.(1996), 243-264; Dimitriu S, editor. “Polysaccharides in medicinalapplication”; New York: Marcel Dekker, pp 575-602. Typically, vaccinesare prepared as injectables, either as liquid solutions or suspensions;solid forms suitable for solution in or suspension in liquid prior toinjection may also be prepared. The preparation may be emulsified or theprotein may be encapsulated in liposomes. The active immunogenicingredients are often mixed with pharmacologically acceptable excipientswhich are compatible with the active ingredient. Suitable excipientsinclude but are not limited to water, saline, dextrose, glycerol,ethanol and the like; combinations of these excipients in variousamounts also may be used. The vaccine may also contain small amounts ofauxiliary substances such as wetting or emulsifying reagents, pHbuffering agents, and/or adjuvants which enhance the effectiveness ofthe vaccine. For example, such adjuvants can include aluminumcompositions, like aluminumhydroxide, aluminumphosphate oraluminumphosphohydroxide (as used in “Gen H-B-Vax®” or “DPT-ImpfstoffBehring”), N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-DMP),N-acetyl-nornuramyl-L-alanyl-D-isoglutamine (CGP 11687, also referred toas nor-MDP),N-acetylmuramyul-L-alanyl-D-isoglutaminyl-L-alanine-2-(1′2′-dipalmitoyl-sn-glycero-3-hydroxyphaosphoryloxy)-ethylamine(CGP 19835A, also referred to as MTP-PE), MF59 and RIBI (MPL+TDM+CWS) ina 2% squalene/Tween-80® emulsion.

The vaccines are usually administered by intravenous or intramuscularinjection. Additional formulations which are suitable for other modes ofadministration include suppositories and, in some cases, oralformulations. For suppositories, traditional binders and carriers mayinclude but are not limited to polyalkylene glycols or triglycerides.Oral formulation include such normally employed excipients as, forexample, pharmaceutical grades of mannitol, lactose, starch, magnesiumstearate, sodium saccharine, cellulose, magnesium carbonate and thelike. These compositions may take the form of solutions, suspensions,tables, pills, capsules, sustained release formulations or powders andcontain about 10% to about 95% of active ingredient, preferably about25% to about 70%.

Vaccines are administered in a way compatible with the dosageformulation, and in such amounts as will be prophylactically and/ortherapeutically effective. The quantity to be administered generally isin the range of about 0.01 μg to 1 g antigen per dose, or about 0.5 μgto 500 μg antigen per dose, or about 1 μg to 300 μg antigen per dose (inthe present case CPS being the antigen), and depends upon the subject tobe dosed, the capacity of the subject's immune system to synthesizeantibodies, and the degree of protection sought. Precise amounts ofactive ingredient required to be administered also may depend upon thejudgment of the practitioner and may be unique to each subject. Thevaccine may be given in a single or multiple dose schedule. A multipledose is one in which a primary course of vaccination may be with one toten separate doses, followed by other doses given at subsequent timeintervals required to maintain and/or to reinforce the immune response,for example, at one to four months for a second dose, and if required bythe individual, a subsequent dose(s) after several months. The dosageregimen also will be determined, at least in part, by the need of theindividual, and be dependent upon the practitioner's judgment. It iscontemplated that the vaccine containing the immunogenic compounds ofthe invention may be administered in conjunction with otherimmunoregulatory agents, for example, with immunoglobulins, withcytokines or with molecules which optimize antigen processing, likelisteriolysin.

For diagnosis and quantification of pathogens like Neisseria, pathogenicfragments, their derivatives, their (poly)peptides (proteins), theirpolynucleotides, etc. in clinical and/or scientific specimens, a varietyof immunological methods, as well as molecular biological methods, likenucleic acid hybridization assays, PCR assays or DNA Enzyme ImmunoAssays (DEIA; Mantero et al., Clinical Chemistry 37 (1991), 422-429)have been developed and are well known in the art. In this context, itshould be noted that the nucleic acid molecules of the invention mayalso comprise PNAs, modified DNA analogs containing amide backbonelinkages. Such PNAs are useful, inter alia, as probes for DNA/RNAhybridization. The proteins of the invention may be, inter alia, usefulfor the detection of anti-pathogenic (like, e.g., anti-bacterial oranti-viral) antibodies in biological test samples of infectedindividuals. It is also contemplated that antibodies of the inventionand compositions comprising such antibodies of the invention may beuseful in discriminating acute from non-acute infections. The CPS asprovided herein can also be used in diagnostic settings, for example as“standards”, in, e.g., chromatographic approaches. Therefore, thepresent CPS can be used in comparative analysis and can be used eitheralone or in combination to diagnostic methods known in the art.

The diagnostic compositions of the invention optionally comprisesuitable means for detection. The CPS as disclosed and described hereinas well as specific antibodies or fragments or derivatives thereofdirected or raised specifically against these CPS are, for example,suitable for use in immunoassays in which they can be utilized in liquidphase or bound to a solid phase carrier. Solid phase carriers are knownto those in the art and may comprise polystyrene beads, latex beads,magnetic beads, colloid metal particles, glass and/or silicon chips andsurfaces, nitrocellulose strips, membranes, sheets, animal red bloodcells, or red blood cell ghosts, duracytes and the walls of wells of areaction tray, plastic tubes or other test tubes. Suitable methods ofimmobilizing nucleic acids, (poly)peptides, proteins, antibodies,microorganisms etc. on solid phases include but are not limited toionic, hydrophobic, covalent interactions and the like. Examples ofimmunoassays which can utilize said proteins, antigenic fragments,fusion proteins, antibodies or fragments or derivatives of saidantibodies of the invention are competitive and non-competitiveimmunoassays in either a direct or indirect format. Commonly useddetection assays can comprise radioisotopic or non-radioisotopicmethods. Examples of such immunoassays are the radioimmunoassay (RIA),the sandwich (immunometric assay) and the Western blot assay.Furthermore, these detection methods comprise, inter alia, IRMA (ImmuneRadioimmunometric Assay), EIA (Enzyme Immuno Assay), ELISA (EnzymeLinked Immuno Assay), FIA (Fluorescent Immuno Assay), and CLIA(Chemioluminescent Immune Assay). Other detection methods that are usedin the art are those that do not utilize tracer molecules. One prototypeof these methods is the agglutination assay, based on the property of agiven molecule to bridge at least two particles.

The CPS of the invention can be bound to many different carriers.Examples of well-known carriers include glass, polystyrene, polyvinylchloride, polypropylene, polyethylene, polycarbonate, dextran, nylon,amyloses, natural and modified celluloses, polyacrylamides, agaroses,and magnetite. The nature of the carrier can be either soluble orinsoluble for the purposes of the invention.

A variety of techniques are available for labeling biomolecules. Thesestechniques are well known to the person skilled in the art and comprise,inter alia, covalent coupling of enzymes or biotinyl groups,iodinations, phosphorylations, biotinylations, random priming,nick-translations, tailing (using terminal transferases) or labeling ofcarbohydrates. Such techniques are, e.g., described in Tijssen,“Practice and theory of enzyme immuno assays”, Burden, R H and vonKnippenburg (Eds), Volume 15 (1985), “Basic methods in molecularbiology”; Davis L G, Dibmer M D; Battey Elsevier (1990), Mayer et al.,(Eds) “Immunochemical methods in cell and molecular biology” AcademicPress, London (1987), or in the series “Methods in Enzymology”, AcademicPress, Inc., or in Fotini N. Lamari, Reinhard Kuhn, Nikos K. Karamanos,“Derivatization of carbohydrates for chromatographic, electrophoreticand mass spectrometric structure analysis”, Journal of Chromatography B,Volume 793, Issue 1, Derivatization of Large Biomolecules, (2003), Pages15-36.

Detection methods comprise, but are not limited to, autoradiography,fluorescence microscopy, direct and indirect enzymatic reactions, etc.

The CPS described herein may be detected by methods known in the art aswell as described and exemplified herein. For example, an ELISA(Enzyme-linked immunosorbent assay) based method described herein may beused for the detection and quantification of the chimeric CPS describedherein. In this context, the chimeric CPS described herein may beimmobilized by an antibody or other binding molecule, such as a lectineor similar, contacting one part or building block of the chimeric CPS.Detection of a second part or building block of the chimeric CPSdescribed herein can be achieved by, e.g., contacting with an antibodyor other binding molecule as described herein which is labeled forfurther detection or a secondary antibody or other binding molecule asdescribed which is labeled for further detection.

As indicated above, the present invention further relates to antibodiesspecifically binding to the synthetic CPS obtainable by the in vitromethods described herein. The term “antibody” herein is used in thebroadest sense and specifically encompasses intact monoclonalantibodies, polyclonal antibodies, multispecific antibodies (e.g.,bispecific antibodies) formed from at least two intact antibodies, andantibody fragments, so long as they exhibit the desired biologicalactivity. Also human, humanized, camelized or CDR-grafted antibodies arecomprised.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies, i.e.the individual antibodies comprising the population are identical exceptfor possible naturally occurring mutations that may be present in minoramounts. Monoclonal antibodies are highly specific, being directedagainst a single antigenic site. Furthermore, in contrast to polyclonalantibody preparations which include different antibodies directedagainst different determinants (epitopes), each monoclonal antibody isdirected against a single determinant on the antigen. In addition totheir specificity, the monoclonal antibodies are advantageous in thatthey may be synthesized uncontaminated by other antibodies. The modifier“monoclonal” indicates the character of the antibody as being obtainedfrom a substantially homogeneous population of antibodies, and is not tobe constructed as requiring production of the antibody by any particularmethod. For example, the monoclonal antibodies to be used in accordancewith the present invention may be made by the hybridoma method firstdescribed by Kohler, G. et al., Nature 256 (1975) 495, or may be made byrecombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567). “Antibodyfragments” comprise a portion of an intact antibody. In context of thisinvention, antibodies specifically recognize CPS obtainable by the invitro method described herein. Antibodies or fragments thereof asdescribed herein may also be used in pharmaceutical and medical settingssuch as vaccination/immunization, particularly passivevaccination/immunization.

The term “antibody” includes antigen-binding portions, i.e., “antigenbinding sites,” (e.g., fragments, subsequences, complementaritydetermining regions (CDRs)) that retain capacity to bind an antigen(such as CPS produced by the herein described methods), comprising oralternatively consisting of, for example, (i) a Fab fragment, amonovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) aF(ab′)2 fragment, a bivalent fragment comprising two Fab fragmentslinked by a disulfide bridge at the hinge region; (iii) a Fd fragmentconsisting of the VH and CH1 domains; (iv) a Fv fragment consisting ofthe VL and VH domains of a single arm of an antibody, (v) a dAb fragment(Ward; 1989; Nature 341; 544-546), which consists of a VH domain; and(vi) an isolated complementarity determining region (CDR). Antibodyfragments or derivatives further comprise F(ab′)2, Fv or scFv fragmentsor single chain antibodies.

The antibodies of the present invention may be used for vaccinatingagainst, treating and/or diagnosing meningococcal meningitidis caused byNeisseria meningitidis serogroup A or X.

The term “carbohydrate” as used herein comprises building blocks such assaccharides and sugars in any form as well as aldehydes and ketones withseveral hydroxyl groups added. A carbohydrate may contain one or more ofsaid building blocks linked via covalent bonds such as glycosidiclinkages. A carbohydrate may be of any length, i.e. it may be monomeric,dimeric, trimeric or multimeric. A carbohydrate may also contain one ormore building blocks as side chains linked to the main chain viacovalent bonds. A carbohydrate may also contain one or more activatedsaccharides such as nucleotide sugars. Examples of nucleotide sugars areUDP-GlcNAc, UDP-ManNAc, UDP-GlcUA, UDP-Xyl, GDP-Man and GDP-Fuc.

Herein, the term “nucleic acid molecule” or “polynucleotide” refers toDNA or RNA or hybrids thereof or any modification thereof that is knownin the state of the art (see, e.g., U.S. Pat. No. 5,525,711, U.S. Pat.No. 4,711,955, U.S. Pat. No. 5,792,608 or EP 302175 for examples ofmodifications). The polynucleotide sequence may be single- ordouble-stranded, linear or circular, natural or synthetic. For instance,the polynucleotide sequence may be genomic DNA, cDNA, mRNA, antisenseRNA, ribozymal or a DNA encoding such RNAs or chimeroplasts (Gamper,Nucleic Acids Research, 2000, 28, 4332-4339). Said polynucleotidesequence may be in the form of a plasmid or of viral DNA or RNA. Forexample, the present invention relates to a nucleic acid molecule havingthe nucleotide sequence of any one of SEQ ID NOs: 1-8, 16-23 and 32. Thepresent invention also encompasses nucleic acid molecules comprising thenucleic acid molecule of any one of SEQ ID NO: 1-8, 16-23 and 32 whereinone, two, three or more nucleotides are added, deleted or substituted.Such a nucleic acid molecule may encode a polypeptide being active (i.e.functional). The term “activity”, “functionality” or “being functional”as used herein refers in particular to the ability of havingsubstantially the same function as the non-modified polynucleotide.

The present invention further relates to nucleic acid molecules whichare complementary to the nucleic acid molecules described above. Alsoencompassed are nucleic acid molecules which are able to hybridize tonucleic acid molecules described herein. A nucleic acid molecule of thepresent invention may also be a fragment of the nucleic acid moleculesdescribed herein. Particularly, such a fragment is a functionalfragment, which means that the fragment has the same function as thenon-modified polynucleotide.

The term “hybridization” or “hybridizes” as used herein in context ofnucleic acid molecules/polynucleotieds/DNA sequences may relate tohybridizations under stringent or non-stringent conditions. If notfurther specified, the conditions are preferably stringent. Saidhybridization conditions may be established according to conventionalprotocols described, for example, in Sambrook, Russell “MolecularCloning, A Laboratory Manual”, Cold Spring Harbor Laboratory, N.Y.(2001); Ausubel, “Current Protocols in Molecular Biology”, GreenPublishing Associates and Wiley Interscience, N.Y. (1989), or Higginsand Hames (Eds.) “Nucleic acid hybridization, a practical approach” IRLPress Oxford, Washington D.C., (1985). The setting of conditions is wellwithin the skill of the artisan and can be determined according toprotocols described in the art. Thus, the detection of only specificallyhybridizing sequences will usually require stringent hybridization andwashing conditions such as 0.1×SSC, 0.1% SDS at 65° C. Non-stringenthybridization conditions for the detection of homologous or not exactlycomplementary sequences may be set at 6×SSC, 1% SDS at 65° C. As is wellknown, the length of the probe and the composition of the nucleic acidto be determined constitute further parameters of the hybridizationconditions. Variations in the above conditions may be accomplishedthrough the inclusion and/or substitution of alternate blocking reagentsused to suppress background in hybridization experiments. Typicalblocking reagents include Denhardt's reagent, BLOTTO, heparin, denaturedsalmon sperm DNA, and commercially available proprietary formulations.The inclusion of specific blocking reagents may require modification ofthe hybridization conditions described above, due to problems withcompatibility.

In accordance to the invention described herein, low stringenthybridization conditions for the detection of homologous or not exactlycomplementary sequences may, for example, be set at 6×SSC, 1% SDS at 65°C. As is well known, the length of the probe and the composition of thenucleic acid to be determined constitute further parameters of thehybridization conditions.

Hybridizing nucleic acid molecules also comprise fragments of the abovedescribed molecules. Furthermore, nucleic acid molecules which hybridizewith any of the aforementioned nucleic acid molecules also includecomplementary fragments, derivatives and allelic variants of thesemolecules. Additionally, a hybridization complex refers to a complexbetween two nucleic acid sequences by virtue of the formation ofhydrogen bonds between complementary G and C bases and betweencomplementary A and T bases; these hydrogen bonds may be furtherstabilized by base stacking interactions. The two complementary nucleicacid sequences hydrogen bond in an antiparallel configuration. Ahybridization complex may be formed in solution (e.g., Cot or Rotanalysis) or between one nucleic acid sequence present in solution andanother nucleic acid sequence immobilized on a solid support (e.g.,membranes, filters, chips, pins or glass slides to which, e.g., cellshave been fixed). The terms complementary or complementarity refer tothe natural binding of polynucleotides under permissive salt andtemperature conditions by base-pairing. For example, the sequence“A-G-T” binds to the complementary sequence “T-C-A”. Complementaritybetween two single-stranded molecules may be “partial”, in which onlysome of the nucleic acids bind, or it may be complete when totalcomplementarity exists between single-stranded molecules. The degree ofcomplementarity between nucleic acid strands has significant effects onthe efficiency and strength of hybridization between nucleic acidstrands. This is of particular importance in amplification reactions,which depend upon binding between nucleic acids strands.

The term “hybridizing sequences” preferably refers to sequences whichdisplay a sequence identity of at least 80%, more preferably at least85%, even more preferably at least 90%, even more preferably at least95%, even more preferably at least 96%, even more preferably at least97%, even more preferably at least 98% and most preferably at least 99%identity with a nucleic acid sequence as described above.

The polypeptides and nucleic acid molecule provided herein preferablyshow a homology, determined by sequence identity, of at least 80%, morepreferably of at least 85%, even more preferably of at least 90%, evenmore preferably of at least 95%, even more preferably of at least 96%,even more preferably of at least 97%, even more preferably of at least98, and most preferably of at least 99% identity to a sequence as shownin any one of SEQ ID NOs: 1-3, 9, 10, 16, 17, 20-25 or 28-33 as definedherein above.

The polypeptides and nucleic acid molecules to be employed in accordancewith this invention preferably show a homology, determined by sequenceidentity, to the sequences indicated under any one of SEQ ID NOs: 1-3,9, 10, 16, 17, 20-25 or 28-33, preferably over the entire length of thesequences compared. The homologous sequences are preferably fragmentshaving a length of at least 100, more preferably at least 200, morepreferably at least 300, more preferably at least 400 and morepreferably at least 500, more preferably at least 600, more preferablyat least 700, more preferably at least 800 and most preferably at least900 nucleotides which have an identity of at least 80%, more preferablyof at least 85%, even more preferably of at least 90%, even morepreferably of at least 95%, even more preferably of at least 96%, evenmore preferably of at least 97%, even more preferably of at least 98,and most preferably of at least 99% with the sequence shown under anyone of SEQ ID NOs: 1-3, 9, 10, 16, 17, 20-25 or 28-33, respectively.

If two sequences which are to be compared with each other differ inlength, sequence identity preferably relates to the percentage of the(amino acid or nucleotide) residues of the shorter sequence which areidentical with the (amino acid or nucleotide) residues of the longersequence. Sequence identity can be determined conventionally with theuse of computer programs such as the Bestfit program (Wisconsin SequenceAnalysis Package, Version 8 for Unix, Genetics Computer Group,University Research Park, 575 Science Drive Madison, Wis. 53711),CLUSTALW computer program (Thompson; 1994; Nucl Acids Res; 2; 4673-4680)or FASTDB (Brutlag; 1990; Comp App Biosci; 6; 237-245). Bestfit utilizesthe local homology algorithm of Smith and Waterman, Advances in AppliedMathematics 2 (1981), 482-489, in order to find the segment having thehighest sequence identity between two sequences. When using Bestfit oranother sequence alignment program to determine whether a particularsequence has for instance 95% identity with a reference sequence of thepresent invention, the parameters are preferably so adjusted that thepercentage of identity is calculated over the entire length of thereference sequence and that homology gaps of up to 5% of the totalnumber of the nucleotides or amino acids in the reference sequence arepermitted. When using Bestfit, the so-called optional parameters arepreferably left at their preset (“default”) values. The deviationsappearing in the comparison between a given sequence and theabove-described sequences of the invention may be caused for instance byaddition, deletion, substitution, insertion or recombination. Such asequence comparison can preferably also be carried out with the program“fasta20u66” (version 2.0u66, September 1998 by William R. Pearson andthe University of Virginia; see also W. R. Pearson, Methods inEnzymology 183 (1990), 63-98, appended examples andhttp://workbench.sdsc.edu/). For this purpose, the “default” parametersettings may be used.

The in vitro methods described herein may also be used for automatedhigh-throughput approaches using robotic liquid dispensing workstations.Generally in such high-throughput approaches a plurality of assaymixtures are run in parallel. These mixtures may have the same ordifferent concentrations of the respective ingredients. Typically, oneof these mixtures serves as a negative control, i.e. at zeroconcentration or below the limits of assay detection.

These and other embodiments are disclosed and obvious to a skilledperson and embraced by the description and the Examples of the presentinvention. Additional literature regarding one of the above-mentionedmethods, means and uses, which can be applied within the meaning of thepresent invention can be obtained from the prior art, for instance inpublic libraries, e.g. with the use of electronic means. For thispurpose, public data bases, such as “Medline”, can be accessed via theinternet, for instance under the addresshttp://www.ncbi.nlm.nih.gov/PubMed/medline.html. Additional data basesand addresses are known to a skilled person and can be taken from theinternet, for instance under the address http://www.lycos.com. Anoverview of sources and information regarding patents or patentapplications in biotechnology is given in Berks, TIBTECH 121 (1994),352-364.

A number of documents including patent applications, manufacturer'smanuals and scientific publications are cited herein. The disclosure ofthese documents, while not considered relevant for the patentability ofthis invention, is herewith incorporated by reference in its entirety.More specifically, all referenced documents are incorporated byreference to the same extent as if each individual document wasspecifically and individually indicated to be incorporated by reference.

The present invention is further described by reference to the followingnon-limiting figures and examples.

The Figures show:

FIG. 1. The donor:acceptor ratio influences the product distribution andthe product length of CsaB- and CsxA-reactions in a different manner

By using CP-A (i.e. CsaB), the product distribution as well as theproduct length can be controlled via the donor:acceptor ratio. By usingfull length CP-X (i.e. CsxA), the product distribution as well as theproduct length cannot be controlled via the donor:acceptor ratio. Heretwo product pools are produced, one having small and the other havinglarge polymers.

A: CPSA distribution obtained after CsaB (in particular ΔN69-CP-A)reactions in the presence of 2 mM UDP-GlcNAc and varying amounts ofshort CPSA oligos (DP1-DP10). It is mentioned that, in pilotexperiments, a similar product length control by varying thedonor-acceptor ratio was seen for the full length CP-A. The designation“expected DP” indicates the major species of the produced capsularpolysaccharides (i.e. the main component) and also specifies thedonor-acceptor ratio (i.e. the donor:acceptor quotient). Thus, theconcentrations of the acceptor CPSX range from 40-4 μM. Accordingly,from left to right, the used ratios of donor carbohydrate to acceptorcarbohydrate are 50:1, 75:1, 100:1, 200:1 and 500:1.

B: Purification of CsxA truncations.

C: Design of CsaB and CsaX truncations. Active truncations are indicatedby and asterisk *.

D: CPSX distribution obtained after CsxA reactions in the presence of 5mM UDP-GlcNAc and varying amounts of short CPSX oligos (DP1-DP7). Thefact that the highest amount of acceptor sugar is not detectable in thegel indicates that all detected molecule are synthesised by CsxA. Themarker consists of polymer of the size which is used in vaccineproduction.

E: CPSX distribution obtained after CsxA reactions in the presence of 5mM UDP-GlcNAc and varying amounts of long CPSX oligos (DP10-DP70). Themarker consists of polymer of the size which is used in vaccineproduction.

FIG. 2. The donor:acceptor ratio influences the product distribution andthe product length of the truncated version of the CsxA

By using a truncated version of CP-X (i.e. dC99-CsxA or dN58dC99-CsxA),the product distribution as well as the product length can be controlledvia the donor:acceptor ratio. The designation “expected DP” indicatesthe major species (i.e. the main component) of the produced capsularpolysaccharides and also specifies the donor-acceptor ratio (i.e. thedonor:acceptor quotient). For example, an “expected DP” of 20, 33, 40,50, 64, 75, 80, 100, 160, 200, 400, or 500 means a ratio of donorcarbohydrate to acceptor carbohydrate of 20:1, 33:1, 40:1, 50:1, 64:1,75:1, 80:1, 100:1, 160:1, 200:1, 400:1, or 500:1.

A/B: CPSX distribution obtained after dN58-CsxA reactions in thepresence of 5 mM UDP-GlcNAc and varying amounts of short CPSX oligos(DP1-DP7).

C/D: CPSX distribution obtained after dC99-CsxA reactions in thepresence of 5 mM UDP-GlcNAc and varying amounts of short CPSX oligos(DP1-DP7).

E: CPSX distribution obtained after dN58dC99-CsxA reactions in thepresence of 5 mM UDP-GlcNAc and varying amounts of short CPSX oligos(DP1-DP7).

F: CPSA distribution obtained after dN69-CsaB reactions in the presenceof 2 mM UDP-GlcNAc and varying amounts of short CPSA oligos (DP1-DP10).

FIG. 3. Determination of the minimal acceptor of full length CsxA andtruncated CsxA

A: Purification of single CPSX oligos.

B-D: Finding the minimal acceptor of CsxA full-length and CsxAtruncations.

FIG. 4. The reaction time influences the product distribution and theproduct length of the truncated versions of the CsxA

By using constant donor-acceptor ratios and truncated versions of theCsxA (i.e. dC99-CsxA, dN58-CsxA or dC99dN58-CsxA) the avDP of theproduced products can be controlled via the reaction time. 50 nM ofcapsule polymerase have been used. The marker indicates length from DP10to DP60.

A: Analysis of the CPSX distribution obtained at different time-pointsduring a CsxA reaction in the presence of 10 mM UDP-GlcNAc and DP7. Theratio of donor carbohydrate to acceptor carbohydrate is approximately8000:1.

B: Analysis of the CPSX distribution obtained at different time-pointsduring a dN58-CsxA reaction in the presence of 10 mM UDP-GlcNAc and DP7.The ratio of donor carbohydrate to acceptor carbohydrate isapproximately 8000:1.

C: Analysis of the CPSX distribution obtained at different time-pointsduring a dC99-CsxA reaction in the presence of 10 mM UDP-GlcNAc and DP7.The ratio of donor carbohydrate to acceptor carbohydrate isapproximately 8000:1.

D: Analysis of the CPSX distribution obtained at different time-pointsduring a dN58dC99-CsxA reaction in the presence of 10 mM UDP-GlcNAc andDP7. The ratio of donor carbohydrate to acceptor carbohydrate isapproximately 8000:1.

E: Analysis of the CPSX distribution obtained at different time-pointsduring a dC99-CsxA reaction in the presence of 10 mM UDP-GlcNAc and DP5.The ratio of donor carbohydrate to acceptor carbohydrate isapproximately 200:1.

F: D: Analysis of the CPSX distribution obtained at differenttime-points during a dN58dC99-CsxA reaction in the presence of 10 mMUDP-GlcNAc and DP7. The ratio of donor carbohydrate to acceptorcarbohydrate is approximately 200:1. A calibration showed that theproduct pool in the 20′ fraction has an avDP of 15 and the product poolin the 20′ fraction has an avDP of 20.

FIG. 5. The oligomerisation status of CP-A (CsaB; upper panel) and CP-X(CsxA; lower panel) was analysed by size exclusion and different saltconcentrations. CP-A elutes in the void volume with 25 mM NaCl andelutes as a broad smeared peak in the presence of 500 mM salt,indicating that the full length enzyme forms aggregates in solution thatare distorted but not dissociated into monomers in the presence of 500mM salt.

In case of CP-X (CsxA) the C-terminal truncation (dC99) is notsufficient to prevent aggregate formation at low salt concentrations,however, the protein migrates with an apparent molecular mass resemblingthe trimer if 500 mM salt are present. In contrast the N-terminaltruncation (dN58) prevents larger aggregate formation. The mutant dN58similar to dN58dC99 migrates with an apparent molecular mass thatsuggest dimer formation already at low salt concentrations (25 mM NaCl).The dimers formed seem to be stable also in the presence of 500 mM salt.

FIG. 6. A: schematic representation of CP-A (upper bar) and CP-X (lowerbar) illustrating the conserved motives existing in these proteins andpositions were point mutations have been introduced. Point mutants withreduced- or no activity are marked with one and double star (*),respectively. B: All protein variants could be expressed as recombinantproteins and purified in high quality. C: A radioactive incorporationassay was used to determine the enzymatic activity.

FIG. 7. Multi-sequence alignment including the bacterial capsulepolymerases that are hexosylphosphate transferases identified a novelconserved asparagine-residue (N324 in CP-X) outside of the conservedmotifs. Based on a hypothetic model of CP-X (top) N324 may be involvedin the coordination of the catalytically important divalent cation(large gray ball in the middle). Replacement of this asparagine byalanine in CP-A and CP-X inactivated enzymatic activity. Remarkably, therespective enzyme isolated from non-pathogenic NmL had no activityneither as wildtype nor after mutation of this position.

FIG. 8. Fluorescently labelled primers for the separate testing ofhexosyl- and sialyltransferase activity.

Starting from 4-MU-Sia, monovalent forms of SiaD_(W-135)/SiaD_(Y) wereused in iterative steps to generate labelled acceptors suited toseparately assay hexosyl- and sialyltransferase activity in the chimericcapsule polymerases of NmW-135 and NmY. Exemplarily shown are theacceptors with Gal as hexose. For simplicity DP—degree ofpolymerization—is used to describe the different 4-MU-labelledsaccharides.

FIG. 9. Chromatographic behaviour of 4-MU-labelled oligomers in anionexchange chromatography.

In consecutive enzymatic reactions 4-MU-Sia (4-MU-DP1) was elongated to4-MU-DP4. Before the start of a new reaction 0.01% of the sample wasloaded onto a CarboPac® PA-100 column and products eluted with a curvedNaNO₂ gradient as shown. MU fluorescence was detected at 375 nm.Overlays of chromatograms obtained with the monovalent forms ofSiaD_(W-135) (A) and SiaD_(Y) (B) are shown. Baseline separation ofpeaks allowed the determination of precise retention times. Of note, thechromatograms show that the conversion of 4-MU-DP1 to 4-MU-DP2 wasincomplete under the conditions used, while all other that reactionsproceeded to completion.

FIG. 10. Time lapse recording of the SiaD_(W-135) reaction.

(A) SiaD_(W-135) was primed with 1 mM 4-MU-Sia-Gal-Sia (4-MU-DP3) in thepresence of 2 mM UDP-Gal and 2 mM CMP-Sia. At indicated time points,product profiles were recorded by HPLC-FD. (B) Enlarged display of theHPLC-FD profile obtained at 40 min and labelling of individual DPs. (C)Normed and weighted peak areas were calculated for each time point andused to construct progress curves. Progress curves obtained in threeindependent experiments (R1-R3) are shown and document the highreproducibility of the reaction. Peaks marked red represent 4-MU-DP1 and4-MU, which represent contaminants in the 4-MU-DP3 pool, whereby 4-MU isalready present in the starting solution of the substrate 4-MU-Sia andrepresents the hydrolysis of the substrate. Both peaks remain unchangedover the entire reaction, confirming a step increase in acceptor qualityfrom DP2 to DP3.

FIG. 11. Use of fluorescent primers to monitor the reaction profiles ofsingle point mutant enzymes

Reaction profiles of the monovalent point mutants NmW-135-(E307A)-His₆and NmW-135-(S972A)-His₆ were recorded in the presence of both donorsugars using the appropriate or false fluorescent primer. Reactions wererun over 30 min with samples taken after 15 sec and 30 min. A:NmW-135-(E307A)-His₆ with 4-MU-DP2. The only reaction product formed is4-MU-DP3. B: NmW-135-(E307A)-His₆ primed with 4-MU-DP3. No product isformed. C: NmW-135-(S972A)-His₆ primed with 4-MU-DP2. No product wasformed. D: NmW-135-(S972A)-His₆ primed with 4-MU-DP3. The only productformed is 4-MU-DP4.

FIG. 12. Schematic representation of the capsule polymerases fromNmW-135 and NmY and illustration of deletion mutants made thereof.

A: Schematic illustration of the full length capsule polymerases NmW-135and NmY. The GT-B folds predicted to comprise hexosyl- andsialyltransferase domain are highlighted in green and purple,respectively. The linker region connecting the GT-B folded domains isshown in black. Mutants made in the course of this project were namedaccording the introduced deletion. All proteins were expressed withN-terminal His₆-tag. B: Western blot analysis of expressed proteins asindicated. Lysates prepared from transformed BL21(DE3) cells wereseparated into soluble and insoluble fraction and after separation on10% SDS-PAGE analysed by western blotting using an anti-penta-Hisantibody.

FIG. 13. Product Profiles of the reactions obtained with CΔ639-His₆ andwith NΔ398-His₆.

Reaction profiles of the truncates variants NmW-135-(CΔ639)-His₆ andNmW-135-(NΔ398)-His₆ were recorded in the presence of both donor sugarsusing the corresponding acceptor. Reactions were run over 30 min withsamples taken after 15 sec and 30 min. A: NmW-135-(CΔ639)-His₆ with4-MU-DP3 catalyze the transfer of UDP-Gal by creating 4-MU-DP4. B:NmW-135-(NΔ398)-His₆ primed with 4-MU-DP2 catalyze the transfer ofCMP-Sia by producing 4-MU-DP3.

FIG. 14. Product profiles of NΔ562 and NΔ609 constructs.

Reaction profiles of the truncates variants NmW-135-(NΔ562)-His₆ andNmW-135-(NΔ609)-His₆ were recorded in the presence of both donor sugarsusing the corresponding acceptor. Reactions were run over 30 min withsamples taken after 15 sec and 30 min. A: NmW-135-(NΔ562)-His₆ with4-MU-DP2 catalyze the transfer of CMP-Sia by creating 4-MU-DP3. B:NmW-135-(NΔ609)-His₆ primed with 4-MU-DP2 catalyze the transfer ofCMP-Sia by producing 4-MU-DP3.

FIG. 15. Quantification of enzymatic activity obtained with mutants.

The enzymatic activity of mutant forms was determined in an HPLC-basedassay using fractions of purified enzymes. The HexTF catalysis is shownunder A which were incubated with 4-MU-DP3 and the SiaTF catalysis isindicated under B which were incubated with 4-MU-DP2. Values representmeans of three independent experiments.

FIG. 16. Secondary structure-based alignment predicted for SiaD_(W-135)and SiaD_(Y).

The sequence alignment was performed using the ClustalW2 alignment andsecondary structure motif predictions The symbols below the sequenceillustrate β-strands (black arrows) and α-helices (lines). Positionswhere truncations were introduced are indicated with arrows above thesequences. The selected sequence accession numbers from the GenBank areY13970 (SiaD_(W-135)) and Y13969 (SiaD_(Y)).

FIG. 17. Mechanisms of polysaccharide synthesis. Polymerization occursby repeated transfer of a sugar residue (Neu5Ac) from a nucleotide sugarprecursor (CMP-Neu5Ac) onto an acceptor substrate (DMB-DP3).

The mechanism of elongation and product distribution are determined bythe nature and extent of interaction with the growing polysaccharidechain. Schematic HPLC profiles of the product distributions are shown atright (DP, degree of polymerization). (a) An extended acceptor bindingsite confers an increased affinity for longer chains which occupy moreof the binding subsites, resulting in uneven chain elongation and askewed product distribution. For highly processive elongation, theacceptor is retained through many transfer cycles and no intermediatesbetween short and long chains will be observed (top, right). Strictnonprocessive elongation involves dissociation of the enzyme-acceptorcomplex after each sugar transfer, therefore, all intermediates can beobserved in the product distribution (bottom, right). In practice, theprecise mechanism is often unclear because the product distributions ofless processive and nonprocessive enzymes are indistinguishable16. (b)In the absence of an extended acceptor binding site, the enzymeinteracts only with the growing end of the polysaccharide, elongation isnonprocessive and distributive, resulting in uniform elongation of allacceptors. The product profile has a narrow, Poissonian distribution(right).

FIG. 18. Drifted polySTs exhibit diverse patterns of chain elongation.

Clones were sorted into three categories based on their average productdispersity. For each category, the reaction time course (top left), HPLCproduct profiles at three reaction time points (bottom), and an extractfrom Supplementary Table 1 (top right) are displayed for fourrepresentative clones. (a) High dispersity clones (mean dispersity >1.2)exhibit an exacerbated kinetic lag phase, maximum reaction rates up to10 times greater than the initial rate, and product profiles stronglyskewed towards longer chains. (b) Medium dispersity clones (meandispersity of 1.1-1.2) exhibit a mild lag phase and less skewed productdistributions. (c) Low dispersity clones (mean dispersity <1.1) have nolag phase (initial rate is the maximum reaction rate) and uniformlyelongate chains resulting in narrow product distributions. Results forall 51 neutral drift clones are given in Supplementary Data Set 2 andSupplementary Table 1.

FIG. 19. Distribution of mutations across the polyST sequence.

Sequencing of 122 ‘neutral’ variants from the first round of screeningrevealed 163 mutations across the Δ25polyST_(NmB) sequence. Here themutations are plotted on an alignment of the four bacterial polySTs fromNeisseria meningitidis serogroup B (NmB), Neisseria meningitidisserogroup C (NmC), Escherichia coli K1 (EcK1) and Escherichia coli K92(EcK92). Similarities of homology are shown in different gray scales.Numbering above the alignment indicates residue number in thepolyST_(NmB) sequence. Of note is that the mutations are unevenlydistributed across the sequence with targeted positions and shortclusters showing high mutability.

FIG. 20. Single amino acid exchanges alter the mode of chain elongation.(a) Reaction time course for single mutatnts which exhibit either anexacerbated (top) or reduced (bottom) kinetic lag phase compared to the_(Δ25)polyST_(NmB) (wild type) reference sequence (dotted line). Errorbars indicate the standard error of three separate enzyme preparations(from different colonies) analysed in a single experiment. (b) Timelapse HPLC product profiles throughout the polymerization reaction ofeach of the clones. The _(Δ25)polyST_(NmB) reference chormatograms (top)are displayed for comparison, and chain lengths are indicated (DP,degree of polymerization; R.T., retention time). Enzymes with anexacerbated lag phase show a broadening of the product distributionspecifically after synthesis of chains >DP10, which corresponds in timeto the increase in reaction rate observed in the reaction time courses.Enzymes with a reduced lag phase show more uniform chain elongation, andconsiderably less broadening of the product distribution.

FIG. 21: Schematic representation of the chromosomal locus (cps) of NmA.Products of genes forming region A are involved in the synthesis of thecapsule polysaccharide and are serogroup-specific. For more information,see text [adapted from Harrison et al. (2013)].

FIG. 22: (A) Coomassie stained SDS-PAGE of purified StrepII-CsaA-His₆(left panel) and CsaC-His₆ (right panel). (B) To select the constructmost suited for the production of active recombinant CsaB the wildtypeand a codon optimized (CsaB_(co)) version of the CsaB sequence werecloned (full length or after N-terminal truncation, Δ69) to produceproteins with tags on either both (StrepII and His₆) or only one end(His₆) as indicated. Transformed bacteria were lysed, separated intosoluble (s) and insoluble (i) fraction and fractions separately run on10% PAGE. After transfer onto nitrocellulose, the blot was developedwith an anti-penta-His antibody. (C) Soluble fractions were used tomeasure CsaB activity in a radioactive incorporation assay. (D)Coomassie stained gel demonstrating the purification result forΔ69-CsaB_(co)-His₆ (IMAC; immobilized metal ion affinity chromatography;SEC size exclusion chromatography).

FIG. 23: Schematic representation of the multi-enzyme assay used tocontinuously follow CsaB activity.

FIG. 24: (A) The chemical properties of the primers used to test theacceptor preference of Δ69-CsaB_(co)-His₆ are displayed. (B)Δ69-CsaB_(co)-His₆ activity was followed using the spectrophotometricassay in the presence of CPSA_(hyd) of avDP6 and avDP15, in eithernative O-acetylated form (CPSA_(hyd(OAc))) or after de-O-acetylation(CPSA_(hyd(deOAc))). Because earlier studies showed that thenon-reducing ends in CPSA_(hyd) are phosphorylated, samples wereadditionally tested before and after phosphatase treatment (deP).Samples designated with _((deOAc))deP were subject of de-acetylation andde-phosphorylation.

FIG. 25: Derivatives of ManNAc ending at the reducing end with a methylgroup (compounds 3, 4) or a phospho-decyl-ester (compounds 1, 2, 5, 6)were synthesized and used to prime the Δ69-CsaB_(co)-His₆ reaction inthe continues spectrophotometric assay. The dimer of ManNAc-1P carryinga phosphodiester at the reducing end was identified as minimal acceptor.Importantly, compound 5 showed acceptor quality identical toCPSA_(hyd(deOAc))-deP and O-acetylation (compound 6) reduced theacceptor quality to approximately the same extend as seen forCPSA_(hyd).

FIG. 26. (A) Products synthesized in the CsaA/CsaB reaction in thepresence of UDP-GlcNAc and CPSA_(hyd) of avDP6 were analysed by highpercentage PAGE and a combined alcian blue/silver staining. Long chainswere produced in the presence of all reactants (reaction) and, though insmall amounts, also in control-1 were no priming oligosaccharides wereadded. The production of long CPSA-chains in control-1 argues for thecapacity of CsaB to start chains de novo. (B) Corresponding ¹H- and ³¹PNMR analysis. (C) HPLC analysis of products obtained in reactions werethe ratio between CsaA:CsaB was varied as indicated. This experimentclearly shows that UDP-formation is a side activity of CsaA, which canbe prevented if the CsaB concentration is equal or higher than theconcentration of CsaA. UMP, UDP and UDP-GlcNAc/UDP-ManNAc were detectedat 280 nm and CPSA at 224 nm.

FIG. 27. (A) In vitro synthesized CPSA (CPSA_(iv)) was separated fromall contaminating reaction products using anion exchange chromatographywith the indicated sodium chloride gradient. (B) Purified CPSAiv afterO-acetylation (CPSA_(iv/OAc) was re-chromatographed under the sameconditions resulting in a well separated product peak, which in dot (C)was recognized by mAb 932. (D) Corresponding ¹H NMR analysis of theproduced CPSA in comparison to CPSA from natural source.

FIG. 28. (A) In vitro synthesis of O-acetylated and non-O-acetylatedCPSA using all enzymes CsaA-C in a one pot reaction. To control productformation, substrates and enzymes were added as indicated. Afterovernight incubation, products were displayed on high percentage PAGE bya combined alcian blue/silver staining. Long chains were synthesized inall reactions containing CsaA, CsaB and UDP-GlcNAc. (B) Productsobtained in the reaction where CsaC and acetyl-CoA were present, weredetected with mAb 932 specifically directed against the CPSA_(OAc).

FIG. 29. Schematic representation of the truncated CsxA constructs. (A)Full-length, (B) ΔN58 truncation, (C) ΔC99 truncation.

FIG. 30. Truncation studies. (A) 10% SDS-PAGE followed by Coomassiestaining of the soluble (LF) and the insoluble (UF) fraction of lysedBL21(DE3)Gold expressing the constructs as indicated. (B) Correspondingwestern blot developed against the His₆-tag. (C) Normalized activitiesfrom soluble fractions of full-length (wildtyp) and truncatedconstructs.

FIG. 31. (A) Analytical size exclusion chromatography in low-salt buffer(25 mM NaCl). (B) Analytical size exclusion chromatography in high-saltbuffer (500 mM NaCl). (C) Schematic representation of the constructstested in A, B and D. (D) Enzymatic activity of the constructs measuredusing and adaption of the multi-enzyme assay published by Freiberger etal. (2007, Molecular Microbiology 65, 1258-1275).

FIG. 32. Elongation of an acceptor carbohydrate having DP5 by using 50nM ΔC99-CP-X (A) or 50 nM ΔN58ΔC99-CP-X (B). The reaction times areindicated at the top. The used marker is avDP15, which is the materialwhich is (coupled to a toxoid) often used in vaccines. The markerindicates length from DP10 to DP60. The donor acceptor ratio isapproximately 200:1. As can be seen ΔN58ΔC99-CP-X (B) produces capsularpolysaccharides with a lower degree of dispersity as compared toΔC99-CP-X (A).

FIG. 33. The capsule polymerase of NmW-135 cannot be regulated via thedonor-acceptor ratio. Extension of the synthetic acceptor4-Mu-Sia-Gal-Sia by CP-W-135 is shown. The concentration of CMP-Sia andUDP-Gal is 2 mM. The designation “ratio” specifies thedonor-acceptor-ratio. Even at a ratio of 4 the synthesized polymers arerather long (approximately >200 repeating units).

FIG. 34. (A) The amount of acceptor was experimentally determined inover-night reactions by incubating ΔN58ΔC99 CsxA in the presence of 10mM UDP-GlcNAc and varying amounts of DP2-DP10. (B) The progress of thereaction could be determined following the consumption of UDP-GlcNAc andthe corresponding production of UMP by HPLC-AEC. (C) The obtained CPSXpool is well in the range of the avDP15 material which is commonly usedfor vaccine production showing that ΔN58ΔC99 CsxA is active and remainsdistributive even if coupled to a solid phase (His-Trap beads). (D) Thefact that ΔN58ΔC99 CsxA can be eluted from the column after the reactionhad been performed demonstrates that the construct was coupled to thesolid phase during the whole period of the reaction.

The Examples illustrate the invention.

EXAMPLE 1 Biochemical Characterisation of the Capsule Polymerases of NmA(CsaB) and NmX (CsxA) Materials and Methods

Freshly transformed E. coli BL21(DE3) were grown at 15° C. in PowerBrothmedium for 18 h. At an optical density of OD₆₀₀=1.0 protein expressionwas induced by addition of 0.1 mM IPTG and allowed to proceed for aperiod of approximately 20 h. Pellets from 125 mL expression culturewere pelleted by centrifugation (6,000×g, 10 min, 4° C.). After awashing step with PBS, cells were re-suspended in 7.5 ml binding buffer(50 mM Tris pH 8.0, 300 mM NaCl) complemented with 40 μg/ml Bestatin(Sigma), 1 μg/ml Pepstatin (Applichem), 100 μM PMSF (Stratagene) andsonified (Branson Digital Sonifier, 50% amplitude, 8×30 s, interruptedby cooling on ice). After centrifugation at 27,000×g for 30 min, thesoluble fractions were directly loaded onto a HisTrap column (GEHealthcare) to enrich the recombinant proteins by immobilized metal ionaffinity chromatography (IMAC). Columns were washed with binding buffer(50 mM Tris pH 8.0, 300 mM NaCl) and proteins eluted in step gradientsusing 10%, 30%, 50% and 100% elution buffer (binding buffer containing500 mM imidazol). Fractions containing recombinant protein were pooledand the buffer exchanged to storage buffer (50 mM Tris pH 8.0, 50 mMNaCl for CsaA/CsaB; 50 mM Hepes pH 7.05, 100 mM NaCl, 5 mM MgCl₂ and 1mM EDTA for CsaC) using the HiPrep 26/10 Desalting column (GEHealthcare). Isolated proteins were concentrated using Amicon Ultracentrifugal devices (Millipore 30 MWCO). After separation into aliquots,samples were snap frozen in liquid nitrogen and stored at −80° C.

For activity testing a radioactive assay system, which is an adaptationof a protocol described by Weisgerber (1990; J. Biol. Chem. 265,1578-1587) was used. Briefly, assays were carried out with 5 μl of thesoluble fractions of bacterial lysates expressing either recombinantCsaB and CsaA or CsxA or the respective purified and epitope-taggedproteins in a total volume of 25 μl assay buffer (50 mM Tris pH 8.0 orvarious pH for determination of the pH optimum). Divalent cations wereadded from stock solutions. The reaction was primed with 5 ng of therespective priming oligosaccharides (hydrolysates of the respectivecapsule polysaccharides) and started by the addition of 0.05 μmolUDP-GlcNAc (Calbiochem) containing 0.05 μCi UDP-[¹⁴C]-GlcNAc (AmericanRadiolabeled Chemicals). Samples were incubated at 37° C. and 5 μlaliquots spotted onto Whatman 3MM CHR paper after 0, 5, 10 and 30 min.Following descending paper chromatography, the chromatographicallyimmobile ¹⁴C-labeled CPSA was quantified by scintillation counting.

Activity Testing of CsaB (Together with CsaA) and CsxA by Use of aMulti-Enzyme Spectrophotometric Assay—

1.2 μM of CsaA and 1 μM CsaB were assayed in the presence of 0.25 mMUDP-GlcNAc (Calbiochem), 20 mM MgCl₂ and 50 mM Tris pH 8.0 in a totalvolume of 100 μL. The consumption of UDP-GlcNAc was coupled tonicotinamide adenine dinucleotide (NADH) consumption using the followingenzymes/substrates: 0.25 mM adenosine triphosphate (ATP, Roche), 1 mMphosphoenolpyruvate (PEP, ABCR), 0.3 mM (NADH, Roche), 9-15 units/mlpyruvate kinase, 13.5-21 units/ml lactic dehydrogenase (PK/LDH mixSigma), 0.05 mg/ml nucleoside monophosphate kinase (Roche). Absorptionwas measured at 340 nm every 30 min using a Biotek EL 808 96-well platereader.

Results The Donor:Acceptor Ratio Influences the Product Distribution ofCsaB- and CsxA-Reactions in a Different Manner

Previous studies have suggested that conjugate vaccines made withintermediate chain-length oligosaccharides, as opposed tohigh-molecular-weight polysaccharides, are more immunogenic and betterat eliciting T cell-dependent antibody responses” (Bröker M, Fantoni S.Minerva Med. (2007) 98(5):575-89). Very effective glycoconjugatevaccines are made of polysaccharides of intermediate size like forexample the quadrivalent glycoconjugate vaccine Menveo (Novartis).

In two recent studies we demonstrated that the capsule polymerases fromNmA (CsaB) and NmX (CsxA) were able to synthesise CPS chains in vitrowhich makes them a very attractive target for in vitro vaccinedevelopment. However, the synthesised chains were very long and thus notready for immediate coupling to the toxin.

With the aim of producing oligosaccharide chains that can be readilycoupled to a carrier protein, we wanted to investigate in this study ifthe product distribution produced by these enzymes can be manipulated.

In a first experiment, we incubated catalytic amounts of the enzymes (50nM) in the presence of different ratios of UDP-GlcNAc, the donor sugar,and a mixture of small oligosaccharides ranging in size from DP1-DP10,the acceptor sugars. The reaction was allowed to proceed over night andthe resulting products were analysed using high percentage PAGE stainedwith a combination of Alcian blue/silver. The expected DPs, indicatedabove the gels (FIG. 1), were calculated from the donor:acceptor ratio.

As a molecular weight marker, we used hydrolysed CPSA and CPSX samplesthat were used in the manufacturing process of Menveo. This way, wecould judge immediately if the produced product pool had the desiredsize.

Indeed, CsaB was able to produce the desired product pool (see FIG. 1A,marker) containing chains ranging from ˜DP20-DP60. Moreover, theobserved chains lengths correlate very well with the expected DPs in therespective reaction. This finding emphasizes that the chain lengthsproduced by CsaB are controlled by the donor:acceptor ratio, indicatinga distributive elongation mechanism. It is of note that the repetitionof the experiment in the presence of an excess of the epimerase CsaAlead to the same results, emphasizing that the observed finding are notdue to limited supply with UDP-ManNAc.

In contrast, we observed a completely different elongation behaviourfrom CsxA (FIG. 1D). At very low donor: acceptor ratios CsxA producedonly very short chains. However, with increasing donor:acceptor ratio weobserved a second product distribution containing chains with a DP muchhigher than expected. The fact that no intermediate chain lengths wereobserved indicates that chains with a certain DP are elongatedpreferably while short chain are disregarded. To test this hypothesis,we repeated the experiment with CPSX oligos ranging from DP10-DP70.Again, the intermediate chain lengths disappeared emphasizing thepossibility that those chains are elongated preferably.

Determination of the Minimal Active Domain of CsaB and CsxA

Both CsxA and CsaB belong to the family of stealth proteins that areinvolved in protecting pathogens from to the host immune defense system(Sperisen, PLoS Comput Biol. (2005) 1(6):e63). All members of stealthharbour, on amino acid level, four conserved regions separated and/orextended by non-conserved regions of varying length. FIG. 1C, showing aschematic alignment of CsxA and CsaB, illustrates that the two enzymesdiffer mostly by the length of their terminal extensions. Thus, wehypothesised that the difference in elongation behaviour might beencoded in the enzymes termini. Example 4 demonstrates that anN-terminal Δ69-truncation of CsaB was active and very well expressed.The experiment shown in FIG. 1A was repeated with this construct and ledto the same results, indicating that the N-terminal 69 aa are notimportant for the product distribution.

Determination of the Minimal Active Domain of CsaB and CsxA

Both CsxA and CsaB belong to the family of stealth proteins (Sperisen etal., 2005). All members of stealth harbour four conserved regions onamino acid level separated and/or extended by non-conserved regions ofvarying length. Based on these sequence characteristics, a schematicalignment of CsaB and CsxA shows that the two enzymes differ mainly intheir terminal extensions (FIG. 1C). Thus, we hypothesised that thedifference in elongation behaviour might be encoded in the enzyme'stermini and designed truncated constructs (FIG. 1C). Starting with CsxA,primers were designed annealing in regions that were predicted to beunstructured by the secondary structure prediction software PHYRE2(Kelley and Sternberg, 2009) to make sure that no important structuralelements were destroyed. Like the full-length enzyme, the CsxAtruncations were expressed as N-terminally MBP- and C-terminallyHis₆-tagged fusion constructs. The radioactive incorporation assay wasused to monitor enzymatic activity from lysates (Fiebig et al., 2014a).While no activity could be detected from constructs lacking CR1(ΔN104-CsxA) and CR4 (ΔC174-CsxA), truncations of the N- (ΔN58-CsxA)and/or C-terminal (ΔC99-CsxA) extensions were active. A combination ofIMAC and SEC was used for the purification of the active constructs.Interestingly, the truncation of the N-terminus led to a nearly six foldincrease of purification yield for ΔN58-CsxA (70 mg/l culture) and a twofold increase for ΔN58ΔC99-CsxA (27 mg/l culture) compared tofull-length-CsxA (12 mg/l culture) or ΔC99-CsxA (13 mg/l culture) andalso reduced the co-purification of degradation products (FIG. 1B). Tocompare the enzymatic activity of the purified constructs tofull-length-CsxA, an adaption of the spectrophotometric assayestablished for CsaB was used (Fiebig et al., 2014b). Interestingly, theactivity of both single truncations was dramatically increased while theactivity of the double truncation was comparable to full-length-CsxA(FIG. 31D).

Analysis of the Oligomerisation State

We recently demonstrated that MBP-CsxA-His₆ forms complexes >669 kDa inthe presence of low amounts of NaCl (25 mM) and partly dissociates intotri- to tetrameric assemblies in high salt conditions (700 mM NaCl)(Fiebig et al., 2014a). The preparative SEC step included in thepurification procedure of the truncated CsxA constructs indicated thatthe termini might influence the oligomerisation behavior of this enzyme.To further interrogate this finding, analytical size exclusionchromatography was performed. ΔC99-CsxA showed salt-dependentoligomerisation behavior similar to full-length-CsxA, suggesting thatthe C-terminus is not involved in the formation of the correctoligomeric state (FIG. 5). However, the truncation of the N-terminusresulted in roughly dimeric assemblies that could be observed at bothhigh and low NaCl concentrations for both ΔN58-CsxA and ΔN58ΔC99-CsxA(FIG. 5).

In contrast to CsxA, no data is available about the oligomerisationstate of CsaB. Since full-length-CsaB is almost exclusively expressed asΔN69-truncation (Fiebig et al., 2014b), we used ΔN69-CsaB for SECanalysis. Like CsxA, this construct also forms high-molecular weightcomplexes >669 kDa in low-salt buffer (FIG. 5). However, at high-saltconditions, no defined oligomerisation state could be detected.

The Termini of CsxA Influence its Elongation Mechanism

Asking if the truncations ΔN58-CsxA, ΔC99-CsxA and ΔN58ΔC99-CsxA showeda difference in their d/a-dependent product profiles, we repeated theexperiment shown in FIG. 1 with these constructs. The product profileobtained for ΔN58-CsxA was comparable to the profile of full-length-CsxA(compare FIGS. 1D and 2A) and a repetition of the experiment atadditional d/a confirmed the appearance of two distinct productpopulations (FIG. 2B). Remarkably, the product profile of ΔC99-CsxA(FIG. 2C) is unlike that of the full-length-CsxA, but resembles that ofthe distributive enzyme CsaB. Combined with testing at further d/a (FIG.2D), these results demonstrate the absence of a processive step in chainelongation, and suggest that processivity is mediated by the truncatedC-terminus of CsxA. Interestingly, this construct shares both elongationbehaviour and its C-terminus, ending directly behind CR4, with the CsaBenzyme (FIG. 1C). In comparison to the product profiles obtained forΔC99-CsxA, ΔN58ΔC99-CsxA requires a far greater d/a to produce productsof a similar size (compare FIGS. 2C and E).

Full-length-CsaB is mostly expressed as ΔN69 truncation with onlyresidual amounts of the original construct present in the purifiedsample (Fiebig et al., 2014b). Consequently, so far, only the productprofile of ΔN69-CsaB has been analysed (FIG. 1A). However, toinvestigate if the minor amounts of full-length-CsaB can influence theproduct profile, we also repeated the experiment shown in FIG. 1A withpurified full-length-CsaB. No influence on the product profile could beobserved (FIG. 2F) indicating that the first 69 amino acids are notimportant for the elongation behaviour of this polymerase.

Determination of the Minimal Acceptor Used by CsaB- and CsxA-Constructs

Protocols in glycoconjugate vaccine manufacturing often require freereducing ends for the conjugation of saccharides to the carrier protein(Costantino et al., 2011). Thus, we wanted to determine the minimallength of CPS acceptors with a free reducing end needed for chainelongation for each of the active constructs. We were especiallyinterested if the d/a-dependent length control of ΔN69-CsaB, ΔC99-CsxAand ΔN58ΔC99-CsxA would be influenced by the DP of the acceptor.Acceptors of a single DP were purified from a mixture of hydrolysed,dephosphorylated CPSX and CPSA using anion exchange chromatography (FIG.3A). The DP of purified material was confirmed by HPAEC-PAD and ¹H NMRusing established protocols (Costantino et al., 1992; Berti et al.,2012). ManNAc-1P could already be excluded as acceptor for CsaB in anearlier study (Fiebig et al., 2014b). Consequently, the acceptor qualityof monomeric compounds (GlcNAc, GlcNAc-1P) was only tested for CsxA.Since both CsaB and CsxA exhibit a de novo polymerisation activity whichis considerably less efficient than elongation of an acceptor, weconsidered acceptors stimulating an increased amount of productsynthesis compared to the de novo reaction to be acceptors for theenzymes.

Full-length-CsxA and the single truncations ΔN58-CsxA and ΔC99-CsxA wereable to elongate acceptors with a DP≧2 while the presence of themonomers GlcNAc-1P and GlcNAc did not lead to product signals beyond denovo background (FIG. 3 C, D, F). Interestingly, a repetition of theexperiment with an acceptor concentration increased by 10-fold showedthat acceptors of a DP≧4 are particularly useful for the d/a-dependentlength-control of ΔC99-CsxA (FIG. 3E). Repeatedly no CPSX wassynthesised by ΔN58ΔC99-CsxA in the presence of DP2 and activity seemedto be impaired also in reactions complemented with DP3 (FIG. 3G). It isof note that the products of ΔN58ΔC99-CsxA were considerably smallerthan those of ΔC99-CsxA under the same reaction conditions (compareFIGS. 3D and G).

For ΔN69-CsaB, a first screening of short CPSA acceptors of a single DPshowed elongation for chains ≧DP2 (FIG. 3I). In agreement with anearlier study suggesting that a phosphate, i.e. a phosphodiester(instead of a methyl-group) at the reducing end is helpful forDP2-uptake (Fiebig et al., 2014b), DP2 with a free reducing end waselongated less efficiently even though HPLC-AEC analysis of DP2 and DP3at 214 nm indicated that the concentration used in the experiment wascomparable for both acceptors. Interestingly, an increase of theacceptor concentration by 25-fold led to an increase in enzymaticactivity in the presence of DP2 (FIG. 3J). However, in agreement withthe observation for ΔC99-CsxA, the best length-control of the productpool was achieved with acceptors of DP≧4 (FIG. 3J) and even after afurther 10-fold increase (compared to the concentrations used in FIG.3J), the size of the product population in the presence of DP2 was notsignificantly reduced (FIG. 3H).

Analysis of the Time-Resolved Elongation Behavior of CsaB and CsxA

To provide further insight into the elongation mechanism used by thedifferent constructs, we analysed the time-course of the polymerasereactions. For this analysis the donor and acceptor concentrations wereincreased to allow detection by HPLC-AEC at 214 nm while maintaining ad/a sufficient for the production of long chains. Samples of eachreaction were heat-inactivated after the indicated time-points andanalyzed via PAGE followed by Alcian-blue/silver staining or HPLC-AEC,which was calibrated using the purified DPs (FIG. 3A).

During the first 60 minutes, full-length-CsxA mainly synthesized shortchains (FIG. 4A) that were at later time-point converted into a broadproduction population consisting of high-molecular weight material.Similar to the full-length construct, ΔN58-CsxA showed thecharacteristics of a processive enzyme and a preference for intermediatechain lengths (FIG. 4B).

The fact that the product profile of ΔC99-CsxA is perfectly controllablevia the d/a suggests distributive elongation (FIG. 2C). However, thisconstruct produces broad product profiles with high dispersity overtime, which is an indication for a certain degree of processivity (FIG.4E). Nevertheless, compared to full-length- and ΔN58-CsxA (FIGS. 4A andB), no gap indicating biased acceptor binding could be detected (FIG. 4Eand FIG. 32 A).

ΔN58ΔC99-CsxA appears to be fully distributive over time and again,produces much shorter chains and narrower distributions compared toΔC99-CsxA (compare FIGS. 4E/F and 32 A/B). However, a repeat of theexperiment with lower acceptor concentrations indicated that thesynthesis of longer chains is possible (FIG. 4D).

Experimental Procedures

General Cloning—

The generation of MBP-csxA-His₆, csaB_(co)-His₆ and d69-csaB_(co)-His₆has been reported elsewhere (Fiebig et al., 2014b; Fiebig et al.,2014a). All truncated constructs described herein were amplified bypolymerase chain reaction (PCR) using the primers shown in Table 1 andgenomic DNA of Nm strain Z2491, csaB_(co) (Fiebig et al., 2014b) orpHC19 (for CsxA sequences)(Fiebig et al., 2014a) as template. Afterrestriction digest using BamHI/XhoI csaB truncations were cloned intopET22b-Strep (Schwarzer et al., 2009) and csxA truncations were clonedinto MBP-csxA-His₆ (T7) or MBP-csxA-His₆ (tac) (Fiebig et al., 2014a).

TABLE 1 Primers used in this study.Restriction sites are highlighted in bold. Primer pairResulting construct GCAGATCTATTGATTTAGT StrepII-ΔN235-CsaB-His₆ATTTACTTGG CCGCTCGAGTTTCTCAAAT GATGATGGTAATG GCAGATCTACTTTAAGTTCStrepII-ΔN167-CsaB-His₆ ATCTATATCT CCGCTCGAGTTTCTCAAAT GATGATGGTAATGGCAGATCTCCTTCTAATCT StrepII-ΔN97-CsaB-His₆ TACTCTTAAGCCCGCTCGAGTTTCTCAAAT GATGATGGTAATG GCAGATCTTTTATACTTAAStrepII-ΔC41-CsaB-His₆ TAACAGAAAATGGC CGCCTCGAGAAAATTCCTT TCTTTAGTTAAGGGCAGATCTTTTATACTTAA StrepII-ΔC25-CsaB-His₆ TAACAGAAAATGGCCGCCTCGAGGTGACTATCA GCACCATCG CGGGATCCCCAATTGAAGA MBP-ΔN58-CsxA-His₆TCCATACCCAGTA CCGCTCGAGTTGTCCACTA GGCTGTGATG GCGGATCCATTATGAGCAAMBP-ΔC99-CsxA-His₆* AATTAGCAAATTG CCGCTCGAGGAGAATTTCT GCTTCTGATACATCCGGGATCCCCAATTGAAGA MBP-ΔN58ΔC99-CsxA-His₆ TCCATACCCAGTACCGCTCGAGGAGAATTTCT GCTTCTGATACATC CGGGATCCGAAAGCACCGAMBP-ΔN104-CsxA-His₆ TATTGCAAGATTCC CCGCTCGAGGAGAATTTCT GCTTCTGATACATCGCGGATCCATTATGAGCAA MBP-ΔC174-CsxA-His₆ AATTAGCAAATTGCCGCTCGAGTTGGCCTGTC AAAATGGCAAAATGGTGG *Leucin 388 of the CsxA sequence,normally encoded by ctt, is encoded by ctc from the XhoI site used forcloning.

Expression and Purification of Recombinant CsaB- and CsxA-Constructs—

Expression and purification of all CsaB- and CsxA-based sequences wasperformed as described in (Fiebig et al., 2014b) and (Fiebig et al.,2014a), respectively. For activity testing from lysates and subsequentanalysis using the radioactive incorporation assay, csxA and itstruncations were expressed from MBP-csxA-His₆ (T7)-based constructsusing BL21(DE3) as expression host (Fiebig et al., 2014a). Purificationwas performed using MBP-csxA-His₆ (tac)-based constructs and M15(pREP4)as expression host.

Activity testing from lysates was performed using the radioactiveincorporation assay as described before (Fiebig et al., 2014b; Fiebig etal., 2014a). The spectrophotometric assay published for CsaB (Fiebig etal., 2014b) was adapted for CsxA resulting in the alterations asfollows; (i) no CsaA (epimerase) was used in the reaction mixture, (ii)the reaction was performed using 54 nM of the respective CsxA constructin the presence of (iii) 37 ng CPSX oligosaccharides of avDP18.6.

SDS-PAGE and Immunoblotting—

SDS-PAGE and immunoblotting was performed as described before (Fiebig etal., 2014b; Fiebig et al., 2014a).

Purification of CPSA and CPSX Oligosaccharides—

CPSA and CPSX oligosaccharide mixtures (Fiebig et al., 2014b; Fiebig etal., 2014a) were dephosphorylated using either acid phosphatase (Sigma)or alkaline phosphatase (CIP, NEB) according to the manufacturer'sguidelines. Anion exchange chromatography (AEC) was performed on anÄKTA_(FPLC) (GE Healthcare) equipped with a MonoQ HR 5/5 column(Pharmacia Biotech) at a flow-rate of 1 ml/min. H₂O and 1 M NaCl wereused as mobile phases M₁ and M₂, respectively. Samples were separatedusing a combination of linear gradients (0% to 5% over 1 ml, 5% to 20%over 10 ml, 20% to 30% over 20 ml). The amount of oligosaccharide ineach fraction was estimated from the peak area obtained at 214 nm underthe assumption that each residue contributes equally to the absorbanceof the respective oligomer. Fractions were dialysed against water(ZelluTrans, Roth, 1 kDa MWCO), freeze-dried and equal concentrationswere adjusted with H₂O. The identity and DP of the oligosaccharidefractions was confirmed by HPLC-PAD and ¹H NMR using establishedprotocols (Berti et al., 2012; Ravenscroft et al., 1999; Costantino etal., 1992).

Analysis of CsaB and CsxA Reaction Products—

For the d/a dependent analysis, 100 nM CsaB and 50 nM CsxA constructswere incubated in 25-100 uL reaction buffer (50 mM Tris pH 8.0, 20 mMMgCl₂, (2 mM DTT, only for CsxA)) in the presence of 2 mM (CsaB) or 5 mM(CsxA) UDP-GlcNAc and acceptors in a concentration resulting in the d/aratios indicated in FIGS. 1 and 4. CsaB reactions were supplemented with0.1 μM (FIG. 1A) or 1 μM (FIG. 4A) CsaA for in situ UDP-ManNAcproduction.

For the time-dependent analysis, the reaction volume was upscaled to 700uL. The UDP-GlcNAc concentration was increased to 10 mM and the CsaBconcentration was decreased to 50 nM. Aliquots were snap-frozen afterthe indicated time-points and heat-inactivated for 3-5 min at 98° C.HPLC-AEC and high percentage PAGE followed by Alcian blue/silverstaining was performed as described before (Fiebig et al., 2014b).

Analytical Size-Exclusion Chromatography—

Roughly 300 ug of recombinant protein was analysed at 280 nm on anÄKTA_(FPLC) (GE Healthcare) equipped with a Superdex 10/300 GL column(GE Healthcare) using 50 mM Tris pH 8.0 elution buffer supplemented withNaCl as indicated in FIG. 3. For the analysis of CsxA constructs, 1 mMof DTT was added. The column was calibrated using the Gel FiltrationMarkers Kit for Protein Molecular Weights 29,000-700,000 Da (Sigma)according to the manufacturer's guidelines.

REFERENCE LIST

-   Berti F, Romano M, Micoli F, Pinto V, Cappelletti E, Gavini M,    Proietti D, Pluschke G, MacLennan C, Costantino P (2012) Relative    stability of meningococcal serogroup A and X polysaccharides.    Vaccine 30: 6409-6415-   Costantino P, Rappuoli R, Berti F (2011) The design of    semi-synthetic and synthetic glycoconjugate vaccines. Expert Opin    Drug Discov 6: 1045-1066-   Costantino P, Viti S, Podda A, Velmonte M, Nencioni L, Rappuoli    R (1992) Development and phase 1 clinical testing of a conjugate    vaccine against meningococcus A and C. Vaccine 10: 691-698-   Fiebig T, Berti F, Freiberger F, Pinto V, Claus H, Romano M R,    Proietti D, Brogioni B, Stummeyer K, Berger M, Vogel U, Costantino    P, Gerardy-Schahn R (2014a) Functional expression of the capsule    polymerase of Neisseria meningitidis serogroup X: a new perspective    for vaccine development. Glycobiology 24: 150-158-   Fiebig T, Freiberger F, Pinto V, Romano M R, Black A, Litschko C,    Bethe A, Yashunsky D, Adamo R, Nikolaev A, Berti F, Gerardy-Schahn R    (2014b) Molecular cloning and functional characterisation of    components of the capsule biosynthesis complex of Neisseria    meningitidis serogroup A: towards in vitro vaccine production. J    Biol Chem-   Kelley L A, Sternberg M J (2009) Protein structure prediction on the    Web: a case study using the Phyre server. Nat Protoc 4: 363-371-   Ravenscroft N, Averani G, Bartoloni A, Berti S, Bigio M, Carinci V,    Costantino P, D'Ascenzi S, Giannozzi A, Norelli F, Pennatini C,    Proietti D, Ceccarini C, Cescutti P (1999) Size determination of    bacterial capsular oligosaccharides used to prepare conjugate    vaccines. Vaccine 17: 2802-2816-   Schwarzer D, Stummeyer K, Haselhorst T, Freiberger F, Rode B, Grove    M, Scheper T, von Itzstein M, Muhlenhoff M, Gerardy-Schahn R (2009)    Proteolytic release of the intramolecular chaperone domain confers    processivity to endosialidase F. J Biol Chem 284: 9465-9474-   Sperisen P, Schmid C D, Bucher P, Zilian O (2005) Stealth proteins:    in silico identification of a novel protein family rendering    bacterial pathogens invisible to host immune defense. PLoS Comput    Biol 1: e63

EXAMPLE 2 Dissection of Hexosyl- and Sialyltransferase-Domain in theBi-Functional Capsule Polymerases from Neisseria meningitidis W-135 andY Provides Evidence for the Existence of a New Sialyltransferase FamilyAbstract

Crucial virulence determinants of disease causing Neisseria meningitidis(Nm) species are their extracellular polysaccharide capsules (CPSs). Inthe serogroups W-135 and Y these are heteropolymers of the repeatingunits [→6)-α-D-Gal-(1-4)-α-Neu5Ac-(2→]_(n) in NmW-135 and[→6)-α-D-Glc-(1→4)-α-Neu5Ac-(2→]_(n) in NmY. We recently showed that thecapsule polymerases, SiaD_(W-135) and SiaD_(Y), which synthesise thesehighly unusual polymers, are composed of two GT-B foldedglycosyltransferase domains (an N-terminal hexosyl- and a C-terminalsialyltransferase domain) and a linker region (amino acids 399-762) ofunknown function. Here we use a mutational approach and syntheticfluorescent substrates to define the boundaries of the hexosyl- andsialyltransferase domains. Our results reveal that the activesialyltransferase domain encompasses a large portion of the linkerregion and indeed may define a new family in the CAZy classification.

Experimental Procedures

Generation of Truncation Mutants and Purification of RecombinantProteins.

To separate hexosyl- and sialyltransferase the clones pHC4(SiaD_(W-135)) and pHC5 (SiaD_(Y)) (Claus (1997) Mol. Gen. Genet. 257,28-34) were used as templates and truncation mutants were generated byPCR as shown in FIG. 12 and FIG. 16. Hot start Phusion-DNA-Polymerase(Thermo Scientific; Fermentas) was used in these experiments primerscontaining NdeI or XhoI sites and PCR conditions were: 1 cycle of 98°C./120 s; 30 cycles 98° C./15 s, 65° C./30 s, 72° C./30 s, and 1 cycle72° C./300 s. Used primers together with the obtained truncationvariants are listed in Table 2. PCR-products after digestion with NdeIand XhoI (New England BioLabs®_(ICn.)) were purified and ligated intothe respective sites of the expression vector pET22-b (Novagen). Aftertransformation into E. coli XL-1 Blue (Stratagene), transformed colonieswhere selected on ampicillin and constructs controlled by restrictionanalysis and sequencing. Expressed proteins carried a C-terminalHis₆-epitope. Purification of recombinant proteins was carried out asdescribed (Romanow, J Biol Chem. (2013) 26; 288(17):11718-30).

TABLE 1 Bacterial strains, plasmids and primers used in this study.Strains/ plasmids/ primers Description or sequence ReferenceE. coli strains BL21(DE3) B; F⁻ompT hsdS_(B)(r_(B) ⁻m_(B)⁻) gal dcm (DE3) Novagen XL1-Blue RecA1 ebdA1 gyrA96 thi-1 hsdR17 supE44Stratagene Plasmids pET-22b(+) Novagen Primers KS422/KS2735′-GCATCTCATATGGCTGTTATTATATTTGTTAACG-3′ wt NmW-1355′-CCGCTCGAGTTTTTCTTGGCCAAAAAACTG-3′ KS422/KS2735′-GCATCTCATATGGCTGTTATTATATTTGTTAACG-3′ wt NmY5′-CCGCTCGAGTTTTTCTTGGCCAAAAAACTG-3′ Point mutants SiaD_(W-135)KS350/KS351 5′-CTGATCATGACATCAGAAAGTGCGGGATTTCCATATATATTTATG-3′ E307A5′-CATAAATATATATGGAAATCCCGCACTTTCTGATGTCATGATCAG-3′ KS370/KS3715′-ATCTCGCGTTGCTGTAGGTGTTTATGCAACTAGCTTATTTG-3′ S972A5′-CAAATAAGCTAGTTGCATAAACACCTACAGCAACGCGAGAT-3′ Point mutants SiaDγAR11/AR12 5′-ATACAGATATCCTAATCATGACATCTCAAAGCGCAGGCTTTGGTTATATAT-3′E307A 5′-ATATATAACCAAAGCCTGCGCTTTGAGATGTCATGATTAGGATATCTGTAT-3′Truncations SiaD_(W-135) KS422/KS4215′-GCATCTCATATGGCTGTTATTATATTTGTTAACG-3′ C-Δ6395′-CCGCTCGAGGCTGCGCGGAAGAATAGTG-3′ AR2/KS2735′-GCATCTCATATGTTTAATAACGTATCATTATCGTC-3′ N-Δ3985-′CCGCTCGAGTTTTTCTTGGCCAAAAAACTG-3′ AR1/KS2735′-GCATCTCATATGACTGATGATAATTTAATACCTAT-3′ N-Δ5625-′CCGCTCGAGTTTTTCTTGGCCAAAAAACTG-3′ AR9/AR2735′-GCATCTCATATGAAATATTCTTATAAATATATCTA-3′ N-Δ6095-′CCGCTCGAGTTTTTCTTGGCCAAAAAACTG-3′ AR10/KS2735′-GCATCTCATATGTCTTGGGAACTTATTCGTGCCTC-3′ N-Δ6395-′CCGCTCGAGTTTTTCTTGGCCAAAAAACTG-3′ KS433/KS2735′-GCATCTCATATGGGTAAGCGTTCGATGGATG-3′ N-Δ6765-′CCGCTCGAGTTTTTCTTGGCCAAAAAACTG-3′ KS434/KS2735′-GCATCTCATATGTCACTGAAAAGTAATGTAGTTG-3′ N-Δ7295-′CCGCTCGAGTTTTTCTTGGCCAAAAAACTG-3′ KS435/KS2735′-GCATCTCATATGAATATCGAAGCATTTCTAAAACC-3′ N-Δ7775-′CCGCTCGAGTTTTTCTTGGCCAAAAAACTG-3′ Truncations SiaDγ KS422/KS4215′-GCATCTCATATGGCTGTTATTATATTTGTTAACG-3′ C-Δ6395′-CCGCTCGAGGCTGCGCGGAAGAATAGTG-3′ KS422/KS3745′-GCATCTCATATGGCTGTTATTATATTTGTTAACG-3′ C-Δ4795′-CCGCTCGAGCGTTTGCATGTTGGGTAAAG-3′ AR2/KS2735′-GCATCTCATATGTTTAATAACGTATCATTATCGTC-3′ N-Δ3985-′CCGCTCGAGTTTTTCTTGGCCAAAAAACTG-3′ AR1/KS2735′-GCATCTCATATGACTGATGATAATTTAATACCTAT-3′ N-Δ5625-′CCGCTCGAGTTTTTCTTGGCCAAAAAACTG-3′ KS433/KS2735′-GCATCTCATATGGGTAAGCGTTCGATGGATG-3′ N-Δ6765-′CCGCTCGAGTTTTTCTTGGCCAAAAAACTG-3′ KS434/KS2735′-GCATCTCATATGTCACTGAAAAGTAATGTAGTTG-3′ N-Δ7295-′CCGCTCGAGTTTTTCTTGGCCAAAAAACTG-3′ Recloning5′-GCATCTCATATGGGTAAGCGTTCGATGGATG-3′ primer5′-CCGCTCGAGTTTTTCTTGGCCAAAAAACTG-3′

Synthesis and Purification of Fluorescently Labelled Acceptors to PrimeSiaD_(W-135) and SiaD_(Y) Reactions.

Because activity testing in the classical radioactive incorporationassay depends on polymer formation (short oligosaccharide primers arewashed out in the paper chromatography step (Weisgerber and Troy (1990)J. Biol. Chem. 265, 1578-1587), the reliable testing of monovalentmutants needed a test system in which single sugar transfers can beidentified. We exploited the capability of SiaD_(W-135) and SiaD_(Y) toextend sialic acid derivatives carrying the fluorescent label2-(4-methylumbelliferyl) (4-MU) at the reducing end. The recombinantC-terminally His₆-tagged monovalent full length enzymesNmW-135-(S972A)-His₆ and NmY-(S972A)-His₆ were used ashexosyltransferases and NmW-135-(E307A)-His₆ and NmY-(E307A)-His₆ assialyltransferases. 4-MU-Sia-Gal/Glc were obtained by mixing 4 mM4-MU-Sia*Na (Sigma or Iris Biotech GmbH) with 4 mM donor sugarUDP-Gal/UDP-Glc (Sigma) and 20 μg ml⁻¹ of the respectivehexosyltransferase (NmW-135-(S972A)-His₆ or NmY-(S972A)-His₆) inreaction buffer (20 mM Tris/HCl pH 8.0, 20 mM MgCl₂, and 2 mM DTT).After 24 h at 25° C. enzymes were removed by ultra-filtration(Amicon®Ultra 10 Molecular Weight Cut-Off (MWCO) Millipore) andfiltrates containing the reaction products (4-MU-Sia-Gal or4-MU-Sia-Glc) were lyophilised (Christ; Alpha 1-2 LD plus). Afterdissolution in water, samples were desalted on P2-gel filtration columns(Bio-Rad). Reaction products were identified by high-performance liquidchromatography using an UFLC-RX system (Shimadzu) coupled to afluorescence detector (RF-10A XL). Samples were excited at 315 nm andmonitored at 375 nm. Although, under the conditions used, this reactiondid not give product yields >90% (see FIG. 9) further productpurification was omitted because the acceptor quality increasedexponential from 4-MU-Sia to 4-MU-Sia-Hex-Sia, making 4-MU-Sia anirrelevant contaminant, which was however useful as an internal standardin consecutive HPLC runs. Obtained 4-MU-Sia-Gal/Glc were then thestarting material for Sia transfer to obtain 4-MU-Sia-Gal/Glc-Sia. Thisreaction was carried out with either NmW-135-(E307A)-His₆ orNmY-(E307A)-His₆ in the presence of 2 mM CMP-Neu5Ac (Nacalai tesque).Reaction conditions were identical to those described in the firstreaction step. However, due to significantly improved acceptor, thesialylation of the starting material was complete after 1 h incubation,after removal of enzyme and desalting, obtained compounds were used initerative rounds to synthesise primers of the needed size.

Enzyme Testing.

Radioactive incorporation assays were performed as described (Romanow, JBiol Chem. (2013) 26; 288(17):11718-30). For activity testing withfluorescent compounds, reactions were carried out in a total volume of25 μl. Mixtures contained 50 mM Tris/HCl pH 8.0, 20 mM MgCl₂, 2 mM DTT,1 mM acceptor (4-MU-Sia-Gal/Glc, 4-MU-Sia-Gal/Glc-Sia, or4-MU-Sia-Gal/Glc-Sia-Gal/Glc) plus 2 mM of the nucleotide sugar(UDP-Gal/Glc; Sigma and/or 2 mM CMP-Neu5Ac; Nacalai tesque) depending onthe tested enzyme. Reactions were started by addition of 20 μg ml⁻¹purified enzyme or, if the soluble fraction of bacterial lysates wasused as enzyme source, with 72-100 μg ml⁻¹ total protein. Afterappropriate incubation times, reactions were stopped by shock freezingin liquid nitrogen.

Synthesised polymers were analysed and quantified via the fluorescenttag. Separation of 4-MU-labelled oligo- and polysaccharides was achievedby anion exchange chromatography (CarboPac® PA-100 column, Dionex) usingan ultrafast HPLC system (UFLC-RX, Shimadzu) with coupled fluorescencedetection (FD; detector RF-10A XL). Before loading onto the CarboPac®PA-100 column, samples were 500-fold diluted in water. For the elutionthe buffer components A (20 mM NaNO₃) and B (1 M NaNO₃) were used toestablish a curved gradient, reaching 21.65% buffer B over 35 min. Theflow rate was set to 0.6 ml min⁻¹ and the column temperature to 50° C.The curved portion of the gradient is described by the following formulain with the index −1.425 describes the slope (LS Solution; Shimadzu;Keys, Glycobiology (2013); 23(5):613-8).

${B\mspace{14mu} \%} = \frac{21.65*( {^{{- 1.425}{t/25}} - 1} )}{( {^{- 1.425} - 1} )}$

Elution profiles were monitored via fluorescence emission at 375 nm with315 nm as extinction wave length. Under these conditions the separationof polysaccharides up to a degree of polymerisation (DP)>18 was easilyachieved (see results).

SDS-PAGE and Immunoblotting.

SDS-PAGE was performed under reducing conditions using 2.5% (v/v)β-mercaptoethanol. Western blot analysis was done on a PVDF membrane(Millipore). For detection of the hexahistidine tag, penta-His antibody(Qiagen) was used as first antibody at a concentration of 1 μg ml⁻¹ anddetected with 0.05 μg ml⁻¹ IgG₁ anti mouse IR Dye800 antibody (OdysseyInfrared Imaging®). Protein bands were visualised and quantified withthe infrared fluorescence detection system (LI-COR®) according to themanufacturer's instructions.

Results

Synthesis of Fluorescently Labelled Oligosaccharides.

With the intention to physically separate hexosyl (Hex)- and sialyl(Sia)-transferase and functionally express the individual enzymedomains, it was prerequisite to have specific acceptors available thatwould allow the unequivocal detection of single sugar transfers. Asdetailed in Experimental Procedures the monovalent mutantsNmW-135-(S972A)-His₆ and NmY-(S972A)-His₆, bearing only HexTF activitywere used to generate 4-MU-Sia-Hex (4-MU-DP2), which then was substratefor the monovalent enzymes bearing only SiaTF activity(NmW-135-(E307A)-His₆ and NmY-(E307A)-His₆). 4-MU-Sia-Hex-Sia (4-MU-DP3)resulting from this reaction, was an efficient acceptor for the HexTFs.The structures of the generated acceptors (exemplarily shown foroligosaccharides with Gal as hexose) are given in FIG. 8.

Based on previous experiences with the separation and purification ofSia-containing oligo- and polymers (Glycobiology (2013) 23(5):613-8) aCarboPac® PA-100 column and a curved NaNO₃ gradient were used todetermine the elution positions of 4-MU-Sia and of the elongatedproducts 4-MU-DP2-4-MU-DP4. Shown in FIGS. 9A and 9B, respectively, arethe products formed with NmW-135-(S972A)-His₆ and NmY-(S972A)-His₆ asHexTFs and NmW-135-(E307A)-His₆ or NmY-(E307A)-His₆ as SiaTFs. Baselineseparation was obtained for all tested compounds. Of note, transfer ofthe neutral sugar (Gal and Glc in the case of NmW-135 and NmY,respectively) shifted the product peaks left in relation to theirprecursors (compare 4-MU-DP2 and 4-MU-DP4 to 4-MU-DP1 and 4-MU-DP3,respectively), indicating that the surface charge density is decisivefor the elution behaviour of the oligosaccharides. The chromatogramsalso show that the first hexosyl-transfer onto 4-MU-DP1 remainedincomplete under the conditions used, while subsequent transferreactions proceeded to completion.

It is important at this point to mention that trials to start thereaction with 4-MU-Gal and 4-MU-Glc as potential substrates for theSiaTF domains were unsuccessful with both, the wild-type enzymes and themonovalent point mutants NmW-135-(E307A)-His₆ and NmY-(E307A)-His₆. Thisfinding strongly suggested that the SiaTF domain needs at least adisaccharide to be active or, alternatively, recognition of the acceptormay need the presence of at least one sialic acid residue.

The new assay system was then used to record the wild-typeSiaD_(W-135/Y) reactions over time. To visualize the initial reactionsteps, the enzymes were primed with 1 mM 4-MU-DP3 in the presence oflimited substrate concentrations (CMP-Sia and UDP-Gal/Glc, 2 mM each)and reactions run up to 40 min. Samples were taken between 0.25 min and40 min as indicated (FIG. 10) and products displayed by HPLC-FD.Exemplarily shown are the results obtained with SiaD_(W-135). Under theconditions used the successive elongation of the 4-MU-DP3 primer up toDP19 (in the case of SiaD_(Y) DP16) could be recorded. The wellseparated peaks migrated at precisely identical positions in threeindependent replicates and thus allowed the assignation of retentiontimes to the individual DP (see also Table 2).

To quantify product formation over time, the relative abundance of eachsynthesized DP at a given time point was calculated and with the help ofthe LC Solution software (Shimadzu) normed and weighted to the totalcurve area giving the normed and weighted values according to theformula:

${{normed}\mspace{14mu} {and}\mspace{14mu} {weighted}\mspace{14mu} {area}} = {\frac{An}{\sum\limits_{n = 1}^{\infty}{An}}*( {n - 1} )}$

with A being the area underneath a single peak and n the number oftransfers needed to form this product. Values were plotted against thereaction time. Data points as shown in (FIG. 10C) are from a singledetermination. The congruence of the curves obtained in threeindependent experiments confirms the reliability of the assay system.

TABLE 2 Degree of synthesised polysaccharides and their correspondingretention time in minutes. CP NmW-135 CP NmY Retention time Degree ofRetention time Degree of [min] Polymerisation [min] Polymerisation 2.308DP₂ 2.381 DP₂ 3.54 DP₁ 3.542 DP₁ 4.487 DP₄ 4.469 DP₄ 5.223 DP₃ 5.184 DP₃6.516 DP₆ 6.4 DP₆ 7.174 DP₅ 7.074 DP₅ 8.197 DP₈ 7.990 DP₈ 8.85 DP₇ 8.633DP₇ 9.623 DP₁₀ 9.315 DP₁₀ 10.257 DP₉ 9.927 DP₉ 10.864 DP₁₂ 10.452 DP₁₂11.47 DP₁₁ 11.047 DP₁₁ 11.965 DP₁₄ 11.461 DP₁₄ 12.544 DP₁₃ 12.026 DP₁₃12.957 DP₁₆ 12.369 DP₁₆ 13.508 DP₁₅ 12.836 DP₁₅ 13.857 DP₁₈ 14.385 DP₁₇14.665 DP₂₀ 15.188 DP₁₉ 15.989 DP₂₁

The illustrated table represents the retention time of appropriateproduced capsule polysaccharides of NmW-135 and NmY during the elutionwith 1 M NaNO₃ using an adapted curve gradient on the CarboPac PA-100column. The elution is based on the charge of oligosaccharides andtherefore all polysaccharides with sialic acid on the reducing end arerepresented by odd numbers and such with hexose on the reducing end arerepresented by even numbers.

Of note, while carbohydrate polymerases normally show a kinetic lagphase (Breyer, Protein Science 10, 1699-1711 (2001)), the progresscurves obtained with SiaD_(W-135/Y) represent typical enzyme reactionkinetic with an initial maximum velocity, indicating that products ofprogressively longer DP are not better acceptors than the startercompound 4-MU-DP3.

As separation of the two active glycosyltranferase domains was the goalof this study, it was important to reconfirm monovalence (Romanow, JBiol Chem. (2013) 26; 288(17):11718-30) of the single point mutantcapsule polymerases NmW-135-(S972A)-His₆ and NmW-135-(E307A)-His₆, usedas positive controls in subsequent experiments. The reaction profilesmonitored in the presence of both donor sugars are shown in FIG. 11.Single transfers onto the acceptors 4-MU-DP2 and 4-MU-DP3 byNmW-135-(E307A)-His₆ and NmW-135-(S972A)-His₆, respectively, (FIG.11A,D) could be shown. Instead, the absence of any visible transfercould be shown for reactions started with primers 4-MU-DP3 and 4-MU-DP2with NmW-135-(E307A)-His₆ and NmW-135-(S972A)-His₆, respectively, (FIG.11B,C) by providing unequivocal proof for the monovalent nature of theseenzymes. Remarkably these pilot experiments in addition showed that Siatransfer onto 4-MU-DP2 occurred very fast. After only 15 sec >70% of theprimer substrate (compare 4-MU-DP2 peak in FIG. 11C) were converted into4-MU-DP3. However, the reaction did not proceed to completion,indicating that and equilibrium between forward and reverse reaction wasreached.

Compared to the sialyltransferase reaction, transfer of Gal onto4-MU-DP3 was slower, with only about 10% of the primer converted to4-MU-DP4 after 15 sec reaction time. However, in contrast to thesialyl-transferase reaction this hexosyltransferase reaction proceededto completeness within 30 min (FIG. 11D). Again, the absence ofadditional reaction products in FIG. 11D and the zero-activity of thecontrol reaction (FIG. 11C) confirmed monovalence of the point mutantenzyme.

Truncation Mutagenesis to Separate Hexosyl- and Sialyltransferase inSiaD_(W-135) and SiaD_(Y).

In FIG. 12A a schematic representation of the linear sequence ofSiaD_(W-135) and SiaD_(Y) is shown. The GT-B folds comprising hexosyl-and sialyltransferase domain are shown separated by a stretch of 379amino acids (dark bar, called linker hereafter). Since in bioinformaticsanalyses neither sequence homologies nor structural folds could beidentified for the linker, we hypothesised that this region is not partof the catalytic domains, but maybe needed to give the GT-B folds thefreedom to tertiary organize. This hypothesis was challenged when theGT-B folds (constructs CΔ639 and NΔ777 see FIG. 12A) were separatelyexpressed and tested in the classical radioactive assay system incomparison to the monovalent enzymes. Though well expressed as solubleproteins (see FIG. 12B) activity was only detectable with thehexosyltransferase domains (NmW-135-CΔ639-His₆ and NmY-CΔ639-His₆) andnot with construct NmW-135-NΔ777-His₆ (FIG. 12A), harboring the putativesialyltransferase of SiaD_(W-135) (data not shown). Because theradioactive assay gave no activity values also with the control enzymes(as outlined in Experimental Procedures, the radioactive assay is notwell suited to analyse the single transfer reactions), the assays werealso carried out with the newly synthesized acceptors 4-MU-DP2 and4-MU-DP3. Now, product profiles obtained with the hexosyltransferasedomains NmW-135-CΔ639-His₆ and NmY-CΔ639-His₆ were similar to thecontrols (see FIG. 11), but again, no activity could be detected withthe isolated sialyltransferase domains (FIG. 13). These result confirmedthat the N-terminal GT-B folds, but not the C-terminal GT-B foldscomprise catalytically active enzymes.

Next, we asked, if the linker (parts of the linker) may be part of theactive sialyltransferase. To test this assumption, mutant NΔ398-His₆,which comprises the entire linker sequence (see FIG. 12A) wasconstructed and was tested directly from bacterial lysates in parallelto the monovalent enzyme (NmW-135-(E307A)-His₆) and the isolated GT-Bfold (NmW-135-CΔ639-His₆). Addition of the linker in constructNΔ398-His₆ restored activity (FIG. 13).

To more precisely define the active sialyltransferase domain additionaltruncations as shown in FIG. 12A were carried out. In this step,attention was given to not hit predicted secondary structure elements(see FIG. 16). Constructs were expressed in E. coli BL21(DE3) bacterialcell line, protein expression monitored by western blotting and activitydetermined with the fluorescently labelled acceptor 4-MU-DP2 in thesoluble fractions of the lysates of transformed bacteria. While theconstructs NΔ562 and NΔ609 showed product profiles similar to thecontrols (FIG. 14) further truncation of 30 amino acids (NΔ639)completely inactivated the sialyltransferase activity. As shown in FIG.16, the cut introduced in NΔ609 is likely to separate a β-strand from apredicted helical stretch, which is missing in NΔ639. The presence ofthis helical element seems of utmost importance not only for theactivity but also for the stability of the truncated protein. WhileNΔ609 is well expressed as a soluble protein, the mutant NΔ639 wasforemost found in the insoluble fraction (FIG. 12B). Further truncationas shown for construct NΔ676, NΔ729, and NΔ777 generated recombinantproteins that were well expressed in the soluble fraction, but had notfunctional activity. This effect on protein stability was confirmed withSiaD_(Y) (see truncations NΔ676 and NΔ729 in FIG. 12B).

Together the mutational studies demonstrated that hexosyl- andsialyltransferase can be separated in SiaD_(W-135) and SiaD_(Y) and canbe expressed as functionally active proteins. Different to theN-terminal GT-B folds, which encode classical hexosyltransferasesbelonging to CAZy family GT-4), the C-terminal GT-B folds comprise onlyparts of the functionally active sialyltransferases. The activesialyltransferases start with a predicted helical segment starting atF609 (see also FIG. 16). The sequence stretch connecting the two GT-Bfolds is thus not just a linking element, but part of the functionalSiaTF domains in SiaD_(W-135/Y). Importantly, our data indicate that thecapsule polymerases of NmW-135 and Y cannot be easily regulated via thedonor-acceptor ratio; see FIG. 33.

The Sialyltransferase Domains in SiaD_(W-135/Y) Define a NewSialyltransferase Family.

As discussed previously (Romanow, J Biol Chem. (2013) 26;288(17):11718-30) Blast search analyses carried out with the C-terminalGT-B folds of SiaD_(W-135) and SiaD_(Y) were unable to assign thesequences to one of the sialyltransferase families existing in the CAZyclassification system (Coutinho (2003) J. Mol. Biol. 328, 307-317).Nevertheless, these searchers revealed weak homology to open readingframes in some bacterial proteins of unknown function. With the currentknowledge that the functional sialyltransferase domains inSiaD_(W-135/Y) encompass the C-terminal part of the linker region (atleast F609-I778), the bioinformatics analyses was repeated and inparallel carried out with the programs BLAST and HMMER. Importantly,HMMER identified more than 30 sequences all comprising bacterialproteins of unknown function. After elimination of identical sequencesfurther analyses by multi-sequence alignments clearly showed highconserved motifs (FIG. 15). The D/E-D/E-G motif, which is part of thecatalytic centre in bacterial sialyltransferases of CAZy families GT-38,GT-52, GT-80 and in pfam 05855 (Freiberger (2007) Molecular Microbiology65, 1258-1275), was found to be replaced by QHG (QYA in SiaD_(W-135/Y)and a sequence encoding a putative sialyltransferase in Listeria). Theknown bacterial Sia-motifs HP and SS/T, that are involved in the bindingof the nucleotide (sugar) (Freiberger (2007) Molecular Microbiology 65,1258-1275; Yamamoto (2007) Biochem. Biophys. Res. Commun 365, 340-343),are highly conserved also in the newly identified sequences but seem tobe part of more extended motifs. Moreover, the new alignment revealed anumber of conserved positions many only present in the SiaD_(W-135/Y)homologues. In summary this alignment suggests the existence of a newsialyltransferase family in which the sialyltransferase domains of thechimeric enzymes SiaD_(W-135/Y) are the only functionally characterizedmembers. No doubt, based on these data, the allocation of SiaD_(W-135/Y)into CAZy family GT-4 needs new consideration.

EXAMPLE 3 Neutral Drift of a Polysialyltransferase Yields Enzymes withProcessive and Distributive Mechanisms

The polysialyltransferase (polyST) from Neisseria meningitidis serogroupB (NmB) synthesises a homopolymer of α2,8-linked sialic acid residuesknown as polysialic acid. The chemical and immunological identity of thebacterial polymer with polysialic acid expressed in the human brainconstitutes a highly effective molecular mimicry which contributes tovirulence of NmB (Muhlenhoff, Curr. Opin. Struct. Biol. 8, 558-564(1998); Roberts, Annu. Rev. Microbiol. 50, 285-315 (1996)). Recentstudies have highlighted the potential of the NmB-polyST forpolysialylation of therapeutic proteins (Lindhout, Proceedings of theNational Academy of Sciences (2011), doi:10.1073/pnas.1019266108), andfor direct therapeutic application of polysialic acid to tissues in vivo(Maarouf, J. Biol. Chem. 287, 32770-32779 (2012)). Despite theimportance of this enzyme, little is known of its structure andfunction.

We have recently developed two new assay systems which enable highthroughput screening and detailed characterisation of polyST activity(Keys, Analytical Biochemistry 427, 107-115 (2012); Keys, AnalyticalBiochemistry 427, 60-68 (2012)). In this study we have used thesemethods to conduct a neutral drift experiment—subjecting the NmB-polySTto high mutation rates followed by purifying selection to removeinactive variants—to explore functional regions of the polyST sequencespace. Detailed analysis of over 50 drifted variants (with 7.3±3.0 aminoacid exchanges per sequence) revealed that some sequences had acquirednew modes of chain elongation which correspond to either a processive ordistributive mechanism of polymerisation. The study identifies sequenceelements which control the mechanism of elongation and the dispersity ofpolymeric products.

The synthesis of uniform glycan structures is a necessary precursor totheir evaluation as therapeutic reagents. We have explored thefunctional sequence space of a bacterial polysialyltransferase andidentified sequence elements controlling the processive or distributivemechanism of chain elongation. The results demonstrate that theseproperties are independent of enzymatic activity and illuminate apathway for the synthesis of uniform oligo- and polysaccharidestructures for research and therapeutic applications.

The extracellular polysaccharides of diverse bacterial species, whichhave emerged from symbiotic or pathogenic coevolution with their humanhosts, are a rich source of structures with important biologicalfunctions and applications as therapeutic reagents (DeAngelis, J.Glycobiology. 23, 764-777 (2013); Pollard Nat. Rev. Immunol. 9, 213-220(2009) and Boltje, Nat. Chem. 1, 611-622 (2009)) The application ofoligo- and polysaccharides will be greatly advanced by strategies tofine tune the functional properties of the polymerizingglycosyltransferases which synthesize these polysaccharides (Boltje,Nat. Chem. 1, 611-622 (2009) and Schmaltz, Chem. Rev. 111, 4259-4307(2011)). Here we demonstrate that these enzymes can readily beengineered to produce uniform Poissonian product distributions.

Our study focused on the polysialyltransferase from Neisseriameningitidis serogroup B (polySTNmB), which synthesizes α2,8-linkedpolysialic acid (polySia), a low abundance polymer with uniquebiological functions and importance as a reagent for research andseveral emerging therapeutic applications. In mammals, polySia is adynamically regulated posttranslational modification predominantly foundon the neural cell adhesion molecule, NCAM (Mühlenhoff, Neurochem. Res.38:1134-1143 (2013)). Due to its polyanionic nature, large size, andwater binding capacity, polySia acts as a powerful anti-adhesive,globally down regulating cellular interactions and increasing cellularmotility (Johnson, J. Biol. Chem. 280, 137-145 (2005)). These uniqueanti-adhesive properties provide plasticity in the developing and adultnervous system (Rutishauser, Nat. Rev. Neurosci. 9, 26-35 (2008)) andplay a role in immune development (Drake, Proc. Natl. Acad. Sci. U.S.A.106, 11995-12000 (2009)). PolySia is re-expressed at the cell surface byvarious tumors to promote invasion and metastatic potential(Hildebrandt, Adv. Exp. Med. Biol. 663, 95-109 (2010)). Moreover, someneuroinvasive bacteria produce an extracellular capsule of polySia whichaids in evasion of the immune system (Corbett, Adv. Appl. Microbiol. 65,1-26 (2008)). PolySia is currently being investigated for diversetherapeutic applications, primarily based on the ability to promoteplastic processes involved in regeneration in neural tissues (ElMaarouf, Adv. Exp. Med. Biol. 663, 137-147 (2010) and El Maarouf, Proc.Natl. Acad. Sci. U.S.A. 103, 16989-16994 (2006)) and its ability toimprove the pharmacological profile of therapeutic proteins decoratedwith polySia (Constantinou, Bioconjugate Chem. 19, 643-650 (2008) andLindhout Proc. Natl. Acad. Sci. U.S.A. 108, 7397-7402 (2011)). A majorstep towards these goals is the demonstration that gene therapy andchemical coupling can be avoided in these applications by using thepolySTNmB to synthesize polySia directly onto therapeutic proteins(Lindhout, Proc. Natl. Acad. Sci. U.S.A. 108, 7397-7402 (2011)),cultured cells, and neural tissues in vivo (Maarouf, J. Biol. Chem. 287,32770-32779 (2012)).

A critical feature of enzymes for use in glycoengineering applicationsis the ability to synthesize uniform products of defined length. Theprimary feature determining product length and dispersity forpolymerases is the extent of their interaction with the growingpolysaccharide acceptor (FIG. 17). An extended binding site confersincreased affinity and an increased rate of transfer to longer chainswhich occupy more of the binding subsites, and may result in processiveelongation (Levengood, J. Am. Chem. Soc. 133, 12758-12766 (2011) andForsee, J. Biol. Chem. 281, 6283-6289 (2006)). This elongation-biascauses broad product distributions which are skewed towards longer chainlengths (FIG. 17a ), and underlies the kinetic lag phase commonlyobserved for polymerizing glycosyltransferases Levengood, M. R., Splain,R. A. & Kiessling, L. L. J. Am. Chem. Soc. 133, 12758-12766 (2011) andKeys Anal. Biochem. 427, 107-115 (2012)). In contrast, the absence ofextended interactions with the polysaccharide acceptor confers adistributive mechanism of elongation, characterized by the absence oflength-bias during elongation, no kinetic lag phase, and uniform productdistributions conforming to a Poisson distribution16 (FIG. 17b ).

With very few exceptions (Jing, J. Biol. Chem. 279, 42345-42349 (2004)),the polymerizing glycosyltransferases which have been characterized todate exhibit an extended acceptor binding site and broad productdistributions—properties which are not amenable to biotechnologicalapplications. The polyST_(NmB) was recently used for enzymaticpolysialylation of therapeutic proteins, however, proteins were modifiedwith a range of chain lengths from 4 to >70 residues at each site(Lindhout, Proc. Natl. Acad. Sci. U.S.A. 108, 7397-7402 (2011)). In thiscase, the assessment of optimal chain length is impossible, and theassessment of the desired modification is weakened due to averaging overmany products. In the ideal case, enzymatic modification would becarried out by perfectly distributive enzymes, resulting in uniformchain elongation and a narrow distribution of product lengths (FIG. 17b). For successful application of polymerizing glycosyltransferases,strategies to engineer the product distribution are urgently needed.

Pioneering studies on ribonuclease A by delCardayré and Raines(delCardayre, Biochemistry 33, 6031-6037 (1994)) demonstrated thatsubstrate binding could be modified independently of catalytic activity,enabling the introduction of processivity without altering activity. Weproposed that this should hold true for polymerizingglycosyltransferases. As no structural data is available to identifyresidues interacting with the acceptor substrate, we conducted a smallneutral drift to explore the polyST_(NmB) sequence space. In a neutraldrift, the target protein is subjected to high mutational loads underselection for the native level of catalytic activity, enabling a broadexploration of functional sequence space and facilitating the emergenceof new properties (Gupta, Nat. Methods 5, 939-942 (2008)).

Our previous experiments have shown that truncation of the N-terminus ofthe polyST_(NmB) increases soluble expression with little cost toenzymatic activity (Keys, Anal. Biochem. 427, 60-68 (2012)). The_(Δ25)polyST_(NmB) was therefore used as the starting point for theneutral drift. In total, three rounds of diversity generation andscreening were conducted. An initial error prone PCR library wasscreened using an in vivo activity assay (Keys, Anal. Biochem. 427,60-68 (2012)) and sequencing of 122 clones revealed 163 ‘neutral’ aminoacid exchanges (FIG. 17). In two subsequent rounds these mutations wereshuffled through the sequence with a high frequency of recombinationwithin short sequence elements and high overall mutation rates (seeMethods). A second tier of in vitro activity screening of a small numberof clones (94 in the second round and 188 in the final screen), using aHPLC based assay (Keys, Anal. Biochem. 427, 107-115 (2012)), ensuredthat the polySTs maintained a level of activity comparable to the_(Δ25)polyST_(NmB) reference sequence. The in vitro activity screen alsoproved to be a bottleneck for genetic diversity with only 21 clonespassing into the third round of diversity generation. However, due tohigh mutagenesis rates, the diversity was sufficient to produce a broadrange of elongation phenotypes. Final screening yielded a pool of 51polymorphic sequences with 7.3±3.0 exchanges per sequence (SupplementaryData Set 1) and a range of activity between approximately 25% and 300%of the reference sequences.

To examine the functional diversity present in the final pool, thereaction time course and pattern of chain elongation was determined forall 51 enzymes. This was achieved using a recently developed fluorescentacceptor, a 1,2-diamino-4,5-methylenedioxybenzene labeled trimer ofsialic acid (DMBDP3), with HPLC separation of reaction products (Keys,Anal. Biochem. 427, 107-115 (2012)) adapted to 96-well format (seeMethods). In order to be able to distinguish different modes ofelongation and make inferences about substrate binding, it is importantthat reactions are carried out with a large substrate to enzyme ratio(Levengood, J. Am. Chem. Soc. 133, 12758-12766 (2011)), therefore anapproximately 100 fold molar excess of the DMB-DP3 acceptor and a 4000fold molar excess of the donor sugar, CMP-Neu5Ac, were used. The enzymesexhibited diverse reaction kinetics and product profiles suggesting thatsequences had indeed drifted in the direction of both processive anddistributive mechanisms of elongation. In order to quantify the tendencyof each enzyme to produce uniform or disperse product profiles, theproduct dispersity (Xw/Xn; where Xw is massaverage DP, and Xn isnumber-average DP) (Stepto, Polym. Int. 59, 23-24 (2010)) wascalculated, and the mean dispersity of products over the reaction coursewas determined for each of the enzymes. The enzymes exhibited a range ofmean dispersity values from 1.06-1.33. As a guide, analysis of the_(Δ25)polyST_(NmB) in the same assay gave a mean dispersity of 1.16, andthe dispersity of a uniform polymer sample with Poisson distribution oflengths approaches 1. The sequences were sorted into three categoriesaccording to high (>1.2), medium (1.1-1.2), and low (<1.1) dispersity.FIG. 18 shows examples of each category which demonstrate the diversemodes of chain elongation within the pool (results for all analyzedclones are given in Table 4).

The enzymes also exhibited different reaction kinetics which correlatedbroadly with the measured product dispersity. Enzymes in the high andmedium dispersity categories displayed skewed product profiles andkinetic lag phases typical of polymerases with extended substratebinding sites (FIG. 18a and b ). In stark contrast are the productprofiles and reaction kinetics of enzymes in the low dispersitycategory. The majority of these enzymes have no lag phase (i.e. initialreaction rate is the maximum observed rate) and uniformly elongateacceptors giving narrow product distributions (FIG. 18c ). For the lowdispersity clones, the product distributions throughout the reactionclosely fit a Poisson distribution, indicating that these enzymes use adistributive mechanism of elongation.

Remarkably, the Lys69Gln mutation is exclusively associated with enzymesin the low dispersity category, which display a distributive mechanismof elongation and Poissonian product distributions. The presence ofnumerous additional mutations in these enzymes indicates that theLys69Gln exchange plays a dominant role in determining the mechanism ofchain elongation. The molecular mechanism by which a single amino acidexchange potently affects chain elongation is of high interest for therational engineering of related polymerases. We speculate that themutation either alters interaction with the growing end of thepolysaccharide or blocks entry of the polysaccharide to the extendedbinding site.

A number of single amino acid exchanges were observed to produce anelongation phenotype. The mutations Arg111His, Lys114Met, Lys143Thr andLys412Ile increase the strength of interaction with chains of DP>10,increasing the observed lag phase and the dispersity of reactionproducts compared to _(Δ25)polyST_(NmB) (FIG. 20). The Lys69Arg,His78Leu and Asn100Ile mutations reduce interaction with the polymer togive a more distributive elongation mechanism (FIG. 20). Interestingly,almost all of the exchanges which were observed to alter the elongationmechanism involve the replacement of large basic amino acids. Togetherthe results indicate that basic residues in the polyST sequencecontribute to an extended interaction interface for binding of thepolyanionic substrate, and that mutations within this site determine themechanism of chain elongation.

Previous neutral drift experiments demonstrated that target enzymes wereable to maintain native levels of activity under high mutational loadsdue to the accumulation of “global suppressor mutations”, whichstabilized the overall protein structure (Gupta, Nat. Methods 5, 939-942(2008)). To test if this held true in our neutral drift, despite theconsiderably smaller screening effort, the most highly accumulatedexchanges within the pool were cloned and examined. Impressively, themajority of exchanges which were highly accumulated during the neutraldrift were observed to increase the yield of activity when present alonein the _(Δ25)polyST_(NmB) sequence (Table 3), and had little or noeffect on product distribution. Notably, the Phe460Ile exchange was themost common mutation, present in 51% of the analyzed clones, and morethan tripled the yield of activity when present alone in the_(Δ25)polyST_(NmB) sequence. The results are consistent with previousneutral drift experiments (Gupta, Nat. Methods 5, 939-942 (2008)), andsuggest that the accumulated mutations may stabilize the polySTstructure.

For the biological function of polymerizing glycosyltransferases it isadvantageous to have extended interaction interfaces which bind andincrease the rate of synthesis of their polysaccharide products.However, for biotechnological applications, the same interfacerepresents a distinct disadvantage, constituting a barrier to thesynthesis of uniform oligo- and polysaccharide structures. The resultsof this study demonstrate that these interaction interfaces can bereadily engineered to yield enzymes with a distributive mechanism andproduct profiles favorable for research and therapeutic applications.

TABLE 3 Single mutations Frequency in Elongation Mean Initial reactionrate* Maximum reaction Mutation drifted clones Phenotype dispersity(pmoles/min) rate* (pmoles/min) K69R n.p. reduced lag 1.143   60 ± 11.296.2 ± 5.4 H78L  9% reduced lag 1.146 121.5 ± 8.5  179.7 ± 11.2 N100I42% reduced lag 1.168   120 ± 22.2 184.1 ± 14.5 R111H 32% exacerbatedlag 1.23 93.2 ± 3.5 274.1 ± 10.5 K114M n.p. exacerbated lag 1.214   92 ±4.3 256.8 ± 2   K143T n.p. exacerbated lag 1.242 56.1 ± 8.9 220.6 ± 10.2I151V n.p. none 1.153 182.2 ± 11.2 329.7 ± 8   K171T 19% none 1.238 37.2± 5.7 150.5 ± 10.9 N180I 13% none 1.21 86.6 ± 6.7 209.8 ± 1.7  I192V 21%none 1.18 135.4 ± 21.7 274.9 ± 18.5 I222V 25% none 1.151 162.8 ± 2.3   278 ± 15.1 I297T 36% none 1.132 197.5 ± 3.3  317.9 ± 14.1 I323V 25%none 1.184 102.6 ± 7.2  222.8 ± 4.6  E368V 32% none 1.213 50.9 ± 3.4140.4 ± 3   T402S 21% none 1.16 133.7 ± 11.2 249.8 ± 11.9 K412I 42%exacerbated lag 1.211 64.4 ± 2.6 237.7 ± 3.9  F460I 51% none 1.194 273.8± 20.6 541.1 ± 16   Δ25polyST_(NmB) 1.181 79.8 ± 7   195.7 ± 1.9  n.pNot present. *Reaction rates are the average ± the standard deviation of3 separate enzyme preparations (from different colonies) in the sameexperiment.

TABLE 4 PolyST clones of the neutral pool Mean Initial reaction Maximumreaction Lag phase Expression Level (% Category Clone dispersity rate(pmoles/min) rate (pmoles/min) (Rate_(max)/Rate_(init)) of solubleprotein) high F154 1.201 57.74 217.11 3.8 1.34 dispersity F117 1.20250.74 136.81 2.7 1.33 F139 1.205 65.38 248.35 3.8 1.71 F123 1.211 50.40168.64 3.3 1.44 F162 1.212 36.57 187.99 5.1 1.33 F164 1.215 28.08 188.626.7 1.30 F072 1.216 131.50 650.61 4.9 1.65 F015 1.219 71.27 366.88 5.11.50 F161 1.221 49.65 186.94 3.8 1.45 F183 1.225 40.65 166.65 4.1 1.41F126 1.236 89.80 233.34 2.6 1.25 F014 1.238 47.55 228.54 4.8 1.79 F1051.239 37.50 198.49 5.3 1.18 F024 1.243 78.18 237.61 3.0 1.84 F180 1.24836.75 191.93 5.2 1.27 F133 1.248 37.04 180.77 4.9 1.09 F102 1.280 69.54449.11 6.5 1.65 F065 1.286 105.99 358.96 3.4 1.61 F093 1.311 61.08261.75 4.3 2.70 F074 1.326 31.06 333.17 10.7 1.40 medium F119 1.12891.88 142.32 1.5 1.13 dispersity F060 1.138 182.61 320.63 1.8 1.72 F0691.148 105.44 220.62 2.1 1.48 F089 1.157 231.48 526.94 2.3 1.51 F1461.168 130.02 258.55 2.0 1.55 F179 1.169 362.79 619.09 1.7 1.75 F1821.170 37.98 105.10 2.8 1.21 F013 1.172 62.31 198.81 3.2 1.40 F043 1.175265.16 419.78 1.6 2.29 F153 1.179 49.34 209.26 4.2 1.23 F103 1.182119.99 324.98 2.7 1.31 F032 1.185 80.91 337.12 4.2 1.76 F175 1.187119.34 348.12 2.9 1.35 F113 1.188 52.10 190.49 3.7 1.26 F036 1.188 53.16193.45 3.6 1.26 F010 1.193 255.71 506.33 2.0 1.79 F159 1.195 94.80214.46 2.3 1.26 F087 1.198 232.60 535.45 2.3 1.84 F185 1.198 113.24278.22 2.5 1.37 low F184 1.060 173.76 173.76 1.0 1.68 dispersity F0781.065 122.02 122.02 1.0 1.79 F079 1.067 205.99 205.99 1.0 1.53 F1161.073 362.27 362.27 1.0 1.47 F034 1.077 79.31 79.31 1.0 1.34 F008 1.07869.62 88.66 1.3 1.50 F129 1.080 111.50 111.50 1.0 1.39 F006 1.085 38.8849.30 1.3 1.66 F138 1.087 152.25 172.60 1.1 1.35 F038 1.089 249.22249.22 1.0 2.16 F028 1.092 76.46 134.30 1.8 1.33 F051 1.095 145.70145.70 1.0 2.40

reference 3775_A 1.160 33.80 134.23 4.0 1.26 sequence 3775_B 1.159 40.65143.34 3.5 1.29 (repeats) 3775_C 1.160 41.42 147.61 3.6 1.32 3775_D1.159 35.09 132.17 3.8 1.31

indicates data missing or illegible when filed

Discussion

Aiming to identify sequence elements contributing to the extendedpolysialic acid binding site in the polyST_(NmB), the in vivo and the invitro assays were combined into a throughput for exploring the polySTsequence space and identifying relationships with the mode of chainelongation. The sequence space was explored using a neutral driftexperiment, where the target sequence is subjected to repeated rounds ofmutagenesis and selection for the native level of activity (Bershtein,J. Mol. Biol. 379, 1029-1044 (2008) and Gupta, Nature Methods 5, 939-942(2008)). Starting from the _(Δ25)polyST_(NmB), three rounds ofmutagenesis and screening were used to generate a pool of 51 polymorphicsequences (7.3±3.0 amino acid exchanges per sequence) with a range ofactivity between approximately 25% and 300% of the original sequence.Each of these clones was then characterised in detail using the DMB-DP3acceptor and HPLC assay. The mutated enzymes exhibited diverse modes ofchain elongation. At one end of the spectrum were clones with productdistributions suggestive of processive elongation. These clones werecharacterised by an exacerbated kinetic lag phase, a strong preferencefor elongated acceptor structures, and highly disperse reactionproducts. At the other end of the spectrum were clones with a highlydistributive mechanism of chain elongation. These were characterised bythe absence of a lag phase (i.e. initial reaction rate was the maximumreaction rate), no preference for elongated acceptors, and uniformproduct distributions.

The diverse elongation phenotypes were associated with specific aminoacid exchanges. Remarkably, the Lys69Gln mutation was exclusivelyassociated with the twelve clones exhibiting a distributive mechanism ofelongation and the most uniform product distributions, indicating adominant role for this residue in determining the mechanism of chainelongation. The Lys69Arg, His78Leu and Asn100Ile mutations also resultedin a narrowing of the product distribution, albeit less striking thanthat of Lys69Gln. In contrast, the mutations Arg111His, Lys114Met,Lys412Ile and Lys143Thr were demonstrated to increase activityspecifically with acceptors of >10 residues in length, causingbroadening of the product distributions.

The fact that most of the exchanges causing an elongation phenotypeinvolve the replacement of basic amino acids suggests that the polyST'sextended binding site is lined with basic residues which interact withthe negative carboxyl groups of the polysialic acid chain. It is typicalof enzymes which act on polymeric substrates to form many weakinteractions with the polymer's repeating structural motifs, whichprevents the formation of strong specific interactions and is thought tofacilitate movement along the polymer (Breyer, Protein Science 10,1699-1711 (2001)). However, during the initial stages of an elongationreaction, such an extended interaction interface confers an increasingaffinity and increasing rate of transfer to chains which occupy more ofthe binding subsites (Levengood, J. Am. Chem. Soc. 133, 12758-12766(2011)). The exact strength and extent of this interaction willsensitively affect the observed product distribution by altering thereaction rate in a chain-length dependent manner. This would explain theimpressive diversity of product distributions in the neutral drift pool.

In contrast, a distributive mechanism of elongation indicates theabsence of extended interactions with the polysaccharide chain andstrictly nonprocessive elongation. Thus, distributive enzymes display nochain-length bias, but uniformly elongate the pool of acceptorsresulting in narrow product distributions which approach a Poissondistribution (Chang, Journal of Molecular Biology 93, 219-235 (1975)),as observed for polySTs containing the Lys69Gln exchange. Theseconsiderations suggest that the Lys69Gln mutation blocks entry of thepolysaccharide to the extended binding site. The proposition thatresidue 69 is involved in acceptor binding is supported by homologymodels of the polyST_(NmB) structure which place this residue on asurface exposed loop in the vicinity of the proposed acceptor bindingsite (F. Freiberger and H. Fuchs, unpublished data).

Importantly, the rapid emergence of different mechanisms of elongation,as observed in this study, indicates that polymerisingglycosyltransferases can be readily engineered to provide desiredproduct distributions. Most promising in this respect is theintroduction of a distributive mechanism with a single amino acidexchange. Distributive enzymes provide uniform (narrow) productdistributions and product length is controlled simply by the ratio ofdonor to acceptor substrates. These are desirable properties forbiotechnological applications where control of polysaccharide length iscritical for the synthesis of defined structures and for the testing ofoptimal chain length for specific applications.

Methods

Molecular Biology.

Plasmids were isolated using NucleoSpin Plasmid™ or Extract™ kits fromMachery-Nagel (Steinheim, Germany). Enzymes were purchased from NewEngland Biolabs (Frankfurt, Germany) and all steps carried out accordingto the manufacturer's instructions. The DNA sequence of the Δ25polySTNmBwas optimized for expression in E. coli. according to guidelines set outby Welch and colleagues24 and synthesized by Eurofins MWG Operon(Ebersberg, Germany). The gene was cloned into pET32a-Strep22 viaBamHI/NotI restriction sites giving the plasmid p3775.

The first library was created by two rounds of error-prone PCR (epPCR),starting on the p3775 template. Buffer condition 3 (320 μM MnSO4; and anadditional 40 μM dGTP) of the Diversify® PCR random mutagenesis kit(Clontec, California, USA) was used with 100 ng of template plasmid in a50 μl reaction using and the T7/T7-term primer pair (Supplementary Table2). PCR amplification was carried out with the following program:

Reaction products were separated on an agarose gel, the amplified genepurified with the NucleoSpin Extract™ kit and eluted in 50 μl. Of theeluted product, 3 μl, was used as template in a second epPCR reactionwith the same buffer conditions and thermocycler program. The product ofthe second reaction was cloned via BamHI/NotI restriction sites into thepET32a-Strep vector22.

The protocol for the second library was designed to facilitate a highlevel of recombination within and between the clusters of highmutability identified from sequencing the first round clones (FIG. 19).Degenerate oligonucleotides encoding all of the diversity observedwithin short sequence elements were designed and ordered fromSigma-Aldrich (Steinheim, Germany) with the highest available purity(HPLC grade). The degenerate oligonucleotides were used to spikestandard gene shuffling reactions with fragments (50-600 bp) of thesequenced first-round clones as template. As the optimal level ofdegenerate oligonucleotide incorporation could not be known, fourreactions with different ratios of [oligonucleotides]:[gene fragments]were used and the reaction products pooled in the final library.

First, 122 plasmids from the first-round clones were pooled and used astemplate for amplification of the coding sequences using the T7/T7-termprimer pair. The amplified product was purified via agarose gelelectrophoresis and 1.6 μg was digested with 0.5 units of DNaseI (NewEngland Biolabs, Frankfurt am Main, Germany) in 40 μl volume for 1 min50 sec at 25° C. The reaction was stopped by addition of 30 mM EDTA andincubation at 75° C. for 10 min. DNaseI products were separated byagarose gel electrophoresis and fragments of 50-600 bp were extractedand purified. Four 30 μl assembly reactions contained either 130 ng, 50ng, 25 ng or 0 ng of the 38 pooled equimolar oligonucleotides(TK229-TK266; Supplementary Table 2), 130 ng of gene fragments, 3% DMSO,200 μM dNTPs, 0.5 units Phusion® DNA polymerase (New England Biolabs,Frankfurt am Main, Germany) and Phusion® HF buffer. The four assemblyreactions were carried out with the following program:

Of each assembly reaction, 5 μl was used as template to amplify fulllength genes in separate PCRs. Four 50 μl amplification PCRs werecarried out with 0.5 units Phusion® DNA polymerase, 6% DMSO, and theTK156/TK157 primer pair (Supplementary Table 2). Reactions werethermocycled according to the following program:

Analysis of 5 μl samples confirmed amplification of similar amounts ofthe full gene in all reactions. Finally all four reactions were pooledand the products cloned as described for the first library. Sequencingof the library pool ensured incorporation of the desired diversity, andsequencing of 12 random clones indicated a mutation rate of 6±5.9 aminoacid exchanges per sequence.

The protocol for the third library was designed to enable fine-gradeshuffling of sequences from the second round of screening with nofurther introduction of mutations. The 22 plasmids from the second-roundclones were pooled, coding sequences were amplified and DNaseI digestedas above, and gene fragments of 50-200 bp were extracted and purified. Asingle PCR assembly and amplification reaction was carried out asdescribed above without the addition of synthetic oligonucleotides. Theshuffled product was cloned into the expression vector as describedabove.

In vivo activity screening was carried out as previously described22.Briefly, library DNA was transformed into the screening strain, MB3109.Transformants were plated on Luria-Bertani (LB) agar at ˜2000 coloniesper 140 mm plate, and grown for 10-12 h at 37° C. until colonies hadreached a diameter of 0.5-1 mm. A nitrocellulose membrane was used tocopy colonies, then placed colonyside-up onto inducing plates. Afterincubation for 3 h at 37° C., colonies were lysed and fixed on themembrane and polySia was detected as previously described22. Positiveclones were picked from the master plates and the amount of polySiasynthesised by each clone was quantified with the same assay inmicrotiter plate format22. In each round of screening 10,000-20,000clones were screened and those producing at least 80% of the wild typelevel of polySia were considered to be neutrally drifting.

Preparation of Recombinant Protein.

Clones for analysis were transformed into BL21-gold(DE3). Colonies werepicked into 150 μl LB media with appropriate antibiotics in 96-well flatbottom plates to allow for observation of optical density (OD) on aplate reader (PowerWave 340, BioTek). Dense overnight cultures werediluted 1:20 into 200 μl PowerBroth (AthenaES, Baltimore, Md., USA) andadjusted to uniform OD600. Cultures were grown at 37° C. untilOD600≈1.8, then cooled on ice and supplemented with isopropylβ-D-1-thiogalactopyranoside (IPTG) to a final concentration of 0.5 mM.Protein expression was carried out at 15° C. for 20 h with shaking at1000 rpm. Cultures typically reached an OD600 of 8.0-9.0. Expressioncultures were harvested in two 100 μl aliquots, pellets were washed withPBS, and stored at −80° C. until analysis.

For cell lysis, pellets were resuspended in 100 μl lysis buffer (50 mMTris pH 8.0, 5% glycerol, 200 μg/ml lysozyme, 10 μg/ml Dnase I, 1 mMPMSF) then subjected to two cycles of freeze-sonication (15 min on ice,snap freezing in liquid nitrogen, and thawing in sonication bath at 23°C.). Unlysed cells and debris were removed by centrifugation at 4000 gfor 10 min, and cleared lysates were supplemented with 1 mM EDTA. Thefinal cell free extracts were used directly in activity assays andprotein determinations.

Polysialyltransferase Assays.

The in vitro activity of polySTs was determined as previouslydescribed18 except that all steps were carried out in 96-well microtiterplates using a 96-channel pipette for accuracy. The recombinant enzymeswere assayed at 25° C. in 100 μl volumes containing 50 mM Tris-HCl pH8.0, 25 mM KCl, 20 mM MgCl2, 5% glycerol, 200-1000 μM CMP-Neu5Ac, and5.32 μM DMBDP3. Reactions were started by the addition of 25 μl of cellfree extract. Reaction samples were quenched by 10-fold dilution in 100mM Tris-HCl pH 8.0, 20 mM EDTA, followed by 10 min at 50° C. Stoppedsamples were centrifuged at 4000 g for 1 h prior to HPLC analysis.Separation of 10 μl samples was carried out on a CarboPac PA-100 columnas previously described18. During the neutral drift experiments eachplate contained two copies of the Δ25polySTNmB reference sequence andclones with at least 25% of the wild type level of activity wereconsidered to be neutrally drifting.

Protein Determinations.

Total protein concentrations were determined with the BCA assay (ThermoScientific, Rockford, Ill., USA) according to the manufacturer'sinstructions.

Determination of polyST expression level was carried out as previouslydescribed22. Briefly, samples of cell free extract were separated by 12%SDS-PAGE. For Western blot analysis, proteins were transferred onto PVDF(polyvinylidene fluoride) membranes (LI-COR Biosciences) and detectedwith 1 μg/ml mouse anti-penta-his antibody (Qiagen) and anti-mouse IR800(1:20,000, LI-COR Biosciences) and quantified by comparison with proteinstandards according to the recommendations of the Odyssey infraredimaging system (LI-COR Biosciences).

EXAMPLE 4 Exploring the Capsule Biosynthesis Machinery of NmA withRegard to its Suitability for In Vitro Vaccine Production Introduction

Neisseria meningitidis serogroup A (NmA) is the major cause ofmeningococcal disease in the African meningitis belt. Besides ofseasonal epidemics that occur with almost annual frequency, NmA has beenthe cause of severe pandemics in the last century (Stephens (2009)Vaccine, 27 Suppl 2, B71-B77). A major virulence factor of Nm is thenegatively charged capsular polysaccharide (CPS). The NmA CPS (CPSA)consists of N-acetyl-mannosamine 1-phosphate units, linked byphosphodiester bonds to give the polymer [→6)-α-D-ManpNAc-(1→OPO₃→]_(n)(Liu (1971) JBC 246 (9), 2849-2858). Of note, the six most virulent Nmserogroups (NmA, -B, -C, -W, -Y and -X) wear negative CPSs. Negativecharge in CPSA and CPSX is due to the phosphodiester group, whilenegative charge in CPSB, -C, -W, and -Y results from the incorporationof the sialic acid (Stephens (2009) Vaccine 27 Suppl 2, B71-B77).

Besides of CPSB, which is identical with polysialic acid in the humanhost, all CPS are immunogenic and cause the production of antibodiesthat are bacteriotoxic in the presence of complement (Gotschlich (1969)The Journal of experimental medicine 129 (6), 1367-1384; Stephens (2007)Lancet 369 (9580), 2196-2210). In fact, this early observation has madethe use of polysaccharide-protein conjugates the gold standard in thedevelopment of vaccines against Nm strains. A number of mono- andtetravalent (the latter comprising serogroups A, C, W and Y) conjugatevaccines against Nm have been licensed (Costantino (2011) Expert opinionon drug discovery 6 (10), 1045-1066).

Crucial to the success of vaccination programs in the sub-Saharanmeningitis belt is the provision of a safe and high-quality vaccine.MenAfriVac®, a conjugate vaccine with CPSA coupled to tetanus toxoid ascarrier protein, has been specifically designed to address these needs(Frasch (2012) Human vaccines & immunotherapeutics 8 (6), 715-724). Withunder 50 cent per dose (Roberts (2010) Science 330 (6010), 1466-1467),mass vaccination campaigns were possible in Burkina Faso, Mali and Nigerand installed herd immunity (Djingarey (2012) Vaccine 30, B40-B45; Caini(2013) Vaccine 31 (12), 1597-1603; LaForce (2011) Eliminating epidemicGroup A meningococcal meningitis in Africa through a new vaccine. Healthaffairs (Project Hope) 30 (6), 1049-1057), protecting not onlyvaccinated but also non-vaccinated individuals and young children(Kristiansen (2013) Clinical infectious diseases 56 (3), 354-363).Recent progress made with the cloning and functional expression ofcapsule polymerases (CPs) (Freiberger (2007) Molecular microbiology 65(5), 1258-1275; Romanow (2013) The Journal of biological chemistry 288(17), 11718-11730; Peterson (2011) Journal of bacteriology 193 (7),1576-1582) and the pioneering studies that demonstrate the suitabilityof the respective recombinant enzymes for the in vitro production ofCPSs (McCarthy (2013) Glycoconjugate journal 30 (9), 857-870; Fiebig(2014) Glycobiology 24:150-8), have opened a new perspective for theeconomic and save production of conjugate vaccines. As NmA is a threadforemost in underdeveloped countries, the development of robust andeconomic regimes for vaccine production is of utmost relevance.Consequently, the goal of the current study was to isolate the capsularpolymerase from NmA and to analyse the capability of the recombinantprotein to produce CPSA in vitro. Because the sugar building blockUDP-ManNAc is commercially not available, it was clear from the start ofthis project that a successful production chain depends on the in situsynthesis of UDP-ManNAc from cheap UDP-GlcNAc. Moreover, as CPSA isimmunogenic only if O-acetylated (Berry (2002) Infection and immunity 70(7), 3707-3713), an effective production chain needs, in addition, theO-acetyltransferase, which is able to perform this modification in anature identical form. Based on these considerations we decided toisolate the three relevant enzymes from NmA and to explore the capacityof the recombinant proteins to work together in vitro to producebio-identical CPSA.

The chromosomal locus (cps for capsular polysaccharide synthesis)contains the genetic information for CPS synthesis, modification andsurface transport. The locus is sub-structured into six regions: A-D, D′and E (FIG. 21). The sequences encoded in region A areserogroup-specific and encode inter alia the polymerases responsible forCPS synthesis (Harrison (2013) Emerging infectious diseases 19 (4),566-573). Regions B and C are highly conserved and encode the proteinsnecessary for export and assembly of the polysaccharide on the cellsurface. In NmA, region A comprises four open reading frames (ORF) csaA,-B, -C and -D (previously designated sacA-D or mynA-D). Usinginsertion-mutagenesis Swartley et al. (1998, Journal of bacteriology 180(6), 1533-1539) demonstrated that each of these genes is involved in theproduction of the NmA capsule. In a later study the gene product encodedin csaC was shown to be an acetyltransferase with specificity for theO-3 and O-4 positions in ManNAc (Gudlavalleti (2004) The Journal ofbiological chemistry 279 (41), 42765-42773). Based on their nucleotideand predicted amino acid sequence, csaA was presumed to encode anUDP-N-acetyl-_(D)-glucosamine 2-epimerase and csaB a capsule polymerase(Swartley (1998) Journal of bacteriology 180 (6), 1533-1539). Additionalevidence that CsaB is in fact the NmA specific capsule polymerase arosefrom the demonstration that it clusters together with CsxA, the provenCP from NmX (Muindi (2014) Glycobiology 24 (2), 139-149; FiebigGlycobiology. (2014) 24:150-8) in the so called stealth protein family,which comprises exclusively D-hexose-1-phosphoryl transferases (Sperisen(2005) PLoS computational biology 1 (6)).

Here we describe the molecular cloning of the genes csaA, csaB and csaCfrom NmA, the production of functional recombinant proteins and provideproof for their functional properties. Using a series of syntheticprimer compounds the ManNAc-dimer linked together by phosphodiesterbonds and carrying a phosphodiester at the reducing end was identifiedto be the minimal acceptor structure, which was preferred by CsaB ifpresented in non-acetylated form. Using a two-step production protocol,O-acetylated CPSA was prepared in highly pure form and was recognized bythe monoclonal antibody (mAb) 935, suggesting that the in vitro producedpolymer represents the bioidentical CPSA.

Results

Cloning and Expression of csaA, csaB and csaC; Production of RecombinantProteins.

Because previous analyses carried out on these genes provided strongevidence that csaA, csaB and csaC encode the UDP-GlcNAc epimerase, thepoly-ManNAc-1-phosphoryl transferase (Swartley (1998) Journal ofbacteriology 180 (6), 1533-1539) and the O-acetyl transferase(Gudlavalleti (2004) The Journal of biological chemistry 279 (41),42765-42773), respectively, primers were constructed (see Material andMethods) to amplify these ORFs. The genomic DNA isolated from Nm strainZ2491 was used as a template. Obtained PCR products were cloned into thepET22b-Strep vector (Schwarzer (2009) The Journal of biologicalchemistry 284 (14), 9465-9474), allowing the expression of recombinantproteins with N-terminal StrepII- and/or C-terminal His₆-tag. Aftertransformation into BL21(DE3) and induction of protein expression (seeMaterial and Methods) the distribution of recombinant proteins betweenthe soluble (s) and insoluble (i) fractions of bacterial lysates wasanalyzed by western blotting against the affinity tags. The recombinantepitope tagged forms of CsaA and CsaC appeared mostly in the solublefraction (data not shown) and could be purified directly from thebacterial lysates. CsaA was purified by IMAC (immobilized metal ionaffinity chromatography) followed by a desalting step and yielded 40 mgprotein/L expression culture. Though some additional faint bands werevisible in Coosmassie stained SDS-PAGE (FIG. 22A), a protein fractionhighly enriched in CsaA was obtained. CsaC was purified following theprotocol described by Gudlavalleti (2004, The Journal of biologicalchemistry 279 (41), 42765-42773) and yielded 96 mg homogenously pureprotein/L culture (FIG. 22A).

Similarly, the StrepII-CsaB-His₆, encoding the putativepoly-ManNAc-1-phosphoryl transferase, was well expressed and appearedwith >60% in the soluble fraction. However, the major product revealedwith the anti-penta-His antibody in western blot migrated with anapparent molecular mass of 50 kDa, strongly deviating from thecalculated molecular mass of 67 kDa (FIG. 22B, left lanes). Becausefaint signals with molecular masses >50 kDa were additionally displayedwith the anti-penta-His antibody, we concluded that StrepII-CsaB-His₆ iseither prone to N-terminal degradation or translated from an alternativestart codon. Consequently, we reinvestigated the NmA genome withbioinformatics techniques. Indeed, two of the used gene predictionsoftwares (GeNmark and GeNmarkS) retrieved an additional ATG (startingwith position 183528 of the NmA genome (NC_003116.1)). In PRODIGAL(Hyatt (2010) BMC bioinformatics 11, 119), the prediction for thissecond start codon was comparable to the published start codon (base No.183321; Parkhill (2000) Nature 404 (6777), 502-506).

To investigate if translation from the alternative ATG leads to a stableprotein, the corresponding truncation Δ69CsaB was cloned with(StrepII-Δ69-CsaB-His₆) and without (Δ69-CsaB-His₆) the N-terminalStrepII-tag. Test expressions in BL21(DE3) demonstrated the occurrenceof proteins of the expected molecular masses, but in repeatedexperiments the level of expressed protein was significantly lower thanfor the full length construct. Moreover, to our surprise, the constructcloned with free N-terminus (Δ69-CsaB-His₆) was routinely higherexpressed than the StrepII-tagged construct (FIG. 22B). Since rarecodons that exist in the CsaB sequence may negatively impact proteinexpression, csaB was codon optimized (using the DNA 2.0 software and thepublished codon frequency tables; Welch et al. (2009)) and expressiontested with the constructs StrepII-CsaB_(co)-His₆ andΔ69-CsaB_(co)-His₆. While increased degradation and concomitantlyreduced expression was seen for StrepII-CsaB_(co)-His₆,Δ69-CsaB_(co)-His₆ was well expressed and no degradation was detectablein western blot with the anti-penta-His antibody (FIG. 22B).

Consecutively, enzymatic activity within the soluble fractions of thebacterial lysates was determined, with a radioactive incorporation assaypreviously developed for the poly-GlcNAc-1-phosphoryl transferase fromNmX (Fiebig, Glycobiology (2014) 24:150-8). The fractions containing therecombinant CsaB variants were tested in the presence of CsaA andUDP-[¹⁴C]GlcNAc. In accordance with the levels of expressed protein(FIG. 22B), StrepII-CsaB-His₆ and Δ69-CsaB_(co)-His₆ showed identicalactivity profiles (FIG. 22C). Based on these results the protein variantΔ69-CsaB_(co)-His₆ was chosen for further experiments. The protein waspurified from the soluble fraction of transformed BL21(DE3) by IMAC andSEC, yielding 60 mg of highly pure protein from 1 L bacterial culture(FIG. 22E).

Optimization of Test Conditions and Determination of CsaB Acceptors.

As the donor sugar (UDP-ManNAc) used by CsaB must be produced in situ inthe epimerase reaction catalyzed by CsaA, the optimization of testconditions needed the presence of both enzymes. As for CPs of other Nmstrains, a hydrolysate of CPSA (CPSA_(hyd)) was used to prime thereaction in the presence of the CsaA substrate UDP-GlcNAc. Initialstudies carried out to evaluate pH and salt conditions showed bestactivity values in the presence of 10-20 mM MgCl₂ and a pH between8.0-8.5 (data not shown). Replacement of Mg²⁺ by Ca²⁺ or Mn²⁺inactivated the enzyme. While these results were similar to what we hadseen with CsxA (CP of NmX), the CsaA/CsaB reaction, in contrast to theCsxA reaction, did not show sensitivity against DTT (up to 2 mM weretested).

The natural CPSA is O-acetylated in positions 3 and 4 of ManNAc (Liu(1971) The Journal of biological chemistry 246 (9), 2849-2858;Gudlavalleti et al., Carbohydr Res. 2006), we therefore interrogated ifΔ69-CsaB_(co)-His₆ recognizes and elongates acetylated andnon-acetylated CPSA_(hyd) with the same efficiency. Therefore, thehydrolysis of CPSA was carried out before and after base treatment toobtain O-acetylated (CPSA_(hyd(OAc))) and de-O-acetylated(CPSA_(hyd(deOAc))) shorter saccharide chains. Knowing that CPSAhydrolysis results in a large distribution of saccharide chain lengths(ranging in size between degree of polymerization, DP, 1 and 70), anionexchange chromatography was used to separate two fractions, the averagedDP (avDP) 6 (comprising DP1-DP10) and avDP15 (comprising DP10-DP70).Both fractions were used to prime the enzyme reactions as indicated inthe subsequent experimental steps.

To quantitatively assess the enzyme reactions, we adapted aspectrophotometric assay previously designed to analyse the CP from NmB(Freiberger (2007) Molecular microbiology 65 (5), 1258-1275). In themulti-enzyme assay shown in FIG. 23, the Δ69-CsaB_(co)-His₆ catalysedproduct formation is coupled to NADH consumption, a step, which can becontinuously followed at 340 nm.

Using this assay the activity of CsaB was determined with CPSA_(hyd)fractions (CPSA_(hyd(OAc)) CPSA_(hyd(deOAc))) of avDP6 and avDP15 (FIG.24). Moreover, because we recently demonstrated that the hydroxyl groupsat position 6 (C₆—OH) on the non-reducing end sugar in CPSA_(hyd) isblocked by phophomonoesters (Ravenscroft (1999) Vaccine 17 (22),2802-2816), both fractions were additionally tested after treatment withacid phosphatase (de-P) to remove this group. Independent of the size ofthe primers used to start the reaction, the native acetylated oligomers(CPSA_(hyd(OAc)) of avDP6 and avDP15 CPSA_(hyd(OAc))) were found to bepoor acceptors, but activity increased steep after removal ofacetyl-groups and even steeper after release of the capping phosphateresidue, making CPSA_(hyd(deOAc))-deP the most efficient acceptors. Thesize of the priming polymers was not of significance for enzymaticactivity (FIG. 24; compare avDP6 and avDP15). The obtained resultsallowed the conclusion that the chain elongation by CsaB proceeds viathe non-reducing end and by transfer of ManNAc-1P onto C₆—OH groups.Furthermore, since CPSA_(hyd(deOAc)) was a better acceptor thanCPSA_(hyd(OAc)), it is likely that O-acetylation takes place on thebuild polymer.

Because no information on the minimal length of the priming acceptor forCsaB could be derived from the CPSA_(hyd) fractions, we used wellcharacterized synthetic compounds to interrogate this question. Thecompounds synthesized are shown in FIG. 25 and varied not only inlength, but also with respect to O-acetylation (compounds 2, 4, 6 were3-O-acetylated) and reducing end modifications. In compounds 1, 2, 5, 6the reducing ends were occupied by a decyl-phosphate-ester, while amethyl group (OMe) was present in the compounds 3 and 4. TheΔ69-CsaB_(co)-His₆ activity did not go beyond background (no acceptor)with compounds 1-4, but, intriguingly, steeply increased withdisaccharides carrying a decyl-phosphate-ester at the reducing end(compounds 5, 6) (FIG. 25). With the non-acetylated compound 5, activityvalues similar to those obtained with the optimized acceptorCPSA_(hyd(deOAc))-deP were measured. In line with the above data (FIG.24), O-acetylation of compound 5 (resulting in compound 6) reduced thequality of the acceptor. Based on these data, the minimal acceptorrecognized by Δ69-CsaB_(co)-His₆ could be defined as the dimer ofManNAc-1P units linked together by phosphodiester bonds. The presence ofa phosphodiester at the reducing end seems obligatory, because compoundsending with OMe groups are not used.

In Vitro Synthesis of CPSA-Chains.

To analyze if long CPSA-chains can be produced with the recombinantenzymes, test reactions were carried out having the enzymesΔ69-CsaB_(co)-His₆ and StrepII-CsaA-His₆ at equal concentration (50 nM)and the priming oligosaccharide (CPSA_(hyd(deOAc))-deP of avDP6) in100-fold molar excess.

Reactions were started by addition of 5 mM UDP-GlcNAc. Control reactions(FIG. 26A control-1-4), in which components were omitted as indicated,were carried out in parallel. After overnight incubation, samples wereloaded onto high percentage PAGE and developed by alcian blue/silverstaining. CPSA_(hyd(deOAc))-deP of avDP15 was loaded as size marker(FIG. 26A). In the presence of CsaA and CsaB the added oligosaccharideprimers were efficiently elongated to long polymer chains (CPSA_(iv),for in vitro produced CPSA). However also in control-1, in the absenceof priming compounds, a faint signal indicating long CPSA was seen. In³¹P NMR (FIG. 26B) the products obtained in the main reaction showed thephosphodiester signal, which is characteristic for CPSA as well as thesignals that indicate the second reaction product UMP. Unexpected werethe signals observed at −5.8 ppm and −9.5 ppm, which indicated theformation of UDP. As these latter signals were most prominent incontrol-4 (FIG. 26 B), with only StrepII-CsaA-His₆ and UDP-GlcNAcpresent, we speculated that UDP is a side product of the epimerasereaction. Support for this assumption was obtained from literature, wereSala et al (1996; Journal of the American Chemical Society 118,3033-3034) had shown that UDP-N-acetylglucosamine 2-epimerase from E.coli, if present at high concentration, accumulates UDP and2-acetamidoglucal, the two intermediates of the epimerization reaction.

Since all relevant ³¹P NMR signals were identified also in the productformed in control-1, this faint CPSA signal demonstrated thatΔ69-CsaB_(co)-His₆ is capable to induce product formation de novo in theabsence of a priming oligosaccharide. Importantly, this same type of denovo synthesis was shown for the CP of NmX (Fiebig, Glycobiology (2014)24:150-8). Still, the self-priming capacity came as a surprise in thecase of CsaB, were de novo formation of product was not seen if thesynthetic compounds 1 or 2 where tested as primers (see FIG. 25).

To reduce UDP-ManNAc hydrolysis and simultaneously complete theincorporation of ManNAc-1P into the product, the ratio between CsaA andCsaB was varied as indicated in FIG. 26C. The reactions carried out asdescribed above with CPSA_(hyd(deOAc))-deP of avDP6 as primer. Afterovernight incubation, products were separated by HPLC and recorded at214 nm (CPSA_(iv)) and 280 nm (UDP and UMP). As long as theconcentration of CsaB was equal or higher than the concentration ofCsaA, no hydrolysis of UDP-ManNAc was detectable (FIG. 26C), even not,if the enzymes were present in only 50 nM concentration and the donorsugar was not completely consumed. These data convincingly show that UDPproduction is a side reaction of the CsaA.

With the intention to produce purified, bio-identical CPSA in milligramamounts, the CsaA/CsaB reaction was up-scaled. The long polymers thatwere obtained in this reaction were purified by anion exchangechromatography (AEC) using a protocol similar to the one described(Fiebig, Glycobiology (2014) 24:150-8). CPSA_(iv) eluted at NaCl clearlyseparated from all other reaction components (FIG. 27A). 1 mg of thepurified CPSA_(iv) was then used for acetylation with the recombinantCsaC. In a second AEC-step CPSA_(iv/OAc) eluted as a single peak (FIG.27B) and the obtained material was recognized by mAB 932 in dot blotanalysis (FIG. 27C). ¹H NMR spectra were recorded for CPSA_(iv) andCPSA_(iv/OAc) and were compared to CPSA from natural source (CPSA_(n))treated (CPSA_(n/deOAc)) or not (CPSA_(n)) with alkaline. The congruenceof obtained spectra confirmed the bioidentity of the synthetic products(FIG. 27D).

Finally we explored at analytical scale, if bio-identically O-acetylatedCPSA can also be produced in the one pot reaction. Therefore, asdescribed above the reaction mixture was supplemented with recombinantCsaC-His₆ and acetyl-CoA. Moreover, control reactions, with singlecompounds missing (see scheme added to FIG. 28) were carried out inparallel. After overnight incubation, products were analyzed byalcian-blue/silver stained high percentage PAGE (FIG. 28A) andimmunoblotting with mAb 932 (FIG. 28B). In the presence of allcomponents, a product recognized by mAb 932 was produced (FIG. 28B),indicating that CsaC-His₆ can acetylate the produced CPSA in situ. Thecontrol reactions carried out in this experiment provided clear evidencefor the functional nature of CsaA and CsaC being UDP-GlcNAc/UDP-ManNAcepimerase and O-acetyl-transferase, respectively. Lastly, the similarity(size and concentration) of reaction products identified in lanes 1, 4,and 6 (FIG. 28A) strongly argue for that suitable conditions wereinstalled for all enzymes in the one-pot reaction scheme.

Discussion

Of all pathogenic Nm serogroups, NmA has caused the most disastrousepidemics in sub-saharan Africa. The prevalence of this pathogen causedan unprecedented attempt in terms of developing a highly effective andeconomic vaccine, MenAfriVac® (Roberts L. Science (2010) 330:1466-7).With coasts of less than 50 cent per dose, MenAfriVac® enabled massvaccination campaigns in Burkina Faso, Mali and Niger (LaForce (2011)Eliminating epidemic Group A meningococcal meningitis in Africa througha new vaccine. Health affairs (Project Hope) 30 (6), 1049-1057;Djingarey (2012) Vaccine 30, B40-B45; Caini (2013) From Agadez toZinder: estimating coverage of the MenAfriVacΓäó conjugate vaccineagainst meningococcal serogroup A in Niger, September 2010-January 2012.Vaccine 31 (12), 1597-1603), which installed herd immunity, leading tothe protection not only for vaccinated but also for non-vaccinatedindividuals and in particular for young children (Kristiansen (2013)Clinical infectious diseases 56 (3), 354-363).

All NmA vaccines licensed today are glycoconjugate vaccines, with CPSAoligosaccharides coupled to carrier proteins. CPSA oligosaccharides arethereby hydrolysis products of CPSA isolated from large scale NmAcultures (Bardotti (2008) Vaccine 26 (18), 2284-2296). To avoid thesignificant coast and biohazard in association with large scale NmAcultures and the pyrogen-free production of polysaccharides, the enzymecatalyzed in vitro synthesis of CPSA would provide an attractivealternative. Towards this goal, we describe in this study, the molecularcloning and functional expression of the three enzymes (UDP-GlcNAc2-epimerase, CsaA; NmA capsule polymerase, CsaB; O-acetyltransferase,CsaC) that are part of the capsular biosynthesis complex in NmA andrepresent the minimal number of enzymes needed to produceimmunologically active CPSA_(OAc) in vitro starting from economicprecursors. Using the well characterized BL21(DE3) strain as expressionhost, C-terminally His₆-tagged versions of CsaA and CsaC could bepurified in high quality and remarkable quantity (CsaA 40 mg and CsaC 96mg per liter bacterial culture).

In CsaB a second start codon was identified starting with methionine-69bp upstream of the first. Use of this second start codon generated astable C-terminally His₆-tagged protein. However, the level of expressedprotein could be significantly increased (to 60 mg/L bacterial culture)after the cDNA sequence was optimized for codon usage in BL21(DE3). Ifthe second start codon is actually used in the natural environmentcannot be answered based on the current data. However, because amulti-sequence alignment demonstrated significant similarity between theN-termini of CsaB and other capsule polymerases starting with the firstmethionine (data not shown), it is likely that the first and not thesecond start codon is used in the natural environment.

Using a two-step protocol (in test reactions even a one pot reaction;see FIG. 28) 0-acetylated CPSA (CPSA_(iv/oAc)) could be produced invitro in high purity and at medium scale. CPSA_(iv/OAc) was of samelength as CPSA isolated from natural source (CPSA_(n)), showed ³¹PNMRand ¹HNMR profiles identical with the natural polymer and was recognizedby mAb 932, a standard reagent in the characterization ofimmunologically active CPSA. Yet it is not proven that acetylationpatters exactly mirror the natural product. However, it likely that alsoNmA glycoconjugate vaccines in which the glycan parts (CPSA_(hyd) ofavDP15) are derived from natural polymers by acidic hydrolysis, showsome variation in the acetylation patterns. Even though this hydrolysisis carried out under optimized conditions, the probability thatacetyl-groups are released is very high.

When the CsaA/CsaB reaction was carried out under suboptimal conditions(meaning that CsaA concentrations were higher than CsaB concentrations)UDP was formed as a side product. Based on control experiments and onearlier literature data the occurrence of UDP indicates that CsaAsimilar to UDP-N-acetylglucosamine 2-epimerase from E. coli, epimerizesUDP-GlcNAc to UDP-ManNAc by forming UDP and 2-acetamidoglucal asintermediate products (Sala (1996) Journal of the American ChemicalSociety 118, 3033-3034). If present in access, the epimerase releasesthese intermediates into solution (Sala Journal of the American ChemicalSociety 118, 3033-3034). Logically, the side reaction was avoided byadjusting the ratio in enzyme concentrations [CsaA]<[CsaB].

O-acetyltransferases from encapsulated bacterial strains have beencloned and functionally characterized (Bergfeld (2007) The Journal ofbiological chemistry 282 (30), 22217-22227). In these studies addressingalso structure-function-relationships the enzymes could be grouped intotwo families. While O-acetyltransferases of NmW-135 and NmY group intothe superfamily of left-handed β-helix (LβH) proteins theO-acetyltransferases isolated from NmA (CsaA) has sequence homology tomembers of the α/β-hydrolase fold family, harboring a catalyticserine-tried which is part of ‘nucleophilic elbow’ motif (Bergfeld(2007) The Journal of biological chemistry 282 (30), 22217-22227;Bergfeld (2009) The Journal of biological chemistry 284 (1), 6-16). Theα/β-hydrolase fold positions one of the catalytic serine-residues to thetip of a narrow turn. This structural motif may impact substraterecognition by the enzyme.

In the current study, we provide clear evidence that the acceptorquality of CPSA_(hyd) increases significantly after release ofO-acetyl-groups. Similarly, the synthetic disaccharide carrying3-O-Ac-groups on ManNAc (compound 6) was a significantly less efficientprimer than the respective compound (5) without O-acetyl-groups. Thesedata argue for that O-acetylation takes place during or after thepolymer is formed. This type of size-dependent substrate recognition hasalready been identified for other O-acetyltransferase that act on sugarpolymers. With CPSA_(hyd) a steep increase in activity was seen afterdephosphorylation of the non-reducing ends. We previously demonstratedthat acidic hydrolysis results (up to 70%) in fragments that arecarrying phosphate at the non-reducing end. Since removal of phosphateimproved the acceptor quality, it can be concluded that chain elongationproceeds by transfer of monosaccharide-phosphates to the non-reducingend.

The use of the synthetic compounds identified two other importantfeatures of CsaB: (i) the minimal efficient acceptor is the dimer and(ii) the reducing end phosphate group can be extended with rather largechemical groups (decyl-ester in the compounds tested in this study).This latter finding bears the perspective that chain elongation can beprimed with reagents of very high purity and functional groups thatfacilitate conjugation of glycans to carrier proteins in the vaccineproduction chain.

Of note is the fact that CsaB is capable of self-priming. The mechanismunderlying the de novo start of chain polymerisation is not understood,but was similarly observed with the CP of NmX (Fiebig, Glycobiology(2014) 24:150-8). In the case of CsaB the interpretation of thephenomenon is however further complicated by the fact that no suchreaction was detectable in the presence of the artificial compounds 1-4.A possible explanation may be that these compounds compete againstUDP-ManNAc in the active site pocket and thus block the initiation ofthe chain. However, more experimental work is needed to understand andexplain this reaction.

Taken together, our study lays a new basis for the development ofefficient and economic protocols with which immunologically active andhighly pure CPSA_(OAc) can be synthesized in vitro and used forglycoconjugate vaccine production. We provide initial evidence thatCPSA_(OAc) can be produced in a one pot reaction and purified tohomogeneity in a single step. The used enzymes are without biohazard andprovide the perspective that production chains can be established alsoin areas that are lacking advanced infrastructural conditions.

Material and Methods

General Cloning—

The genomic DNA isolated from Nm strain Z2491 was a kind gift from Dr.Heike Claus (Institute for Hygiene and Microbiology, University ofWürzburg). The csaB sequence was codon optimized for use in E. coliBL21(DE3) using the Gene Designer software package (DNA 2.0) (Villalobos(2006) BMC bioinformatics 7, 285) and the codon frequency tablespublished by Welch (2009; PloS ONE 4 (9)). The mean codon frequency foreach amino acid was calculated from the codon frequency tables FreqA andFreqB (Welch (2009) PloS ONE 4 (9)) and the resulting codon frequencytable used as template for the in silico generation of csaB_(co).csaB_(co) flanked 5′ by a BamHI site and 3′ by a XhoI site wassynthesized from Eurofins MWG Operon. All other csaA-C sequencesdescribed herein were amplified by polymerase chain reaction (PCR) usingthe primers shown in Table 4 and genomic DNA from Nm strain Z2491 orcsaB_(co) as template. PCR products were cloned via the restrictionsites shown in Table 4 into the corresponding sites of the vectorpET22b-Strep (Schwarzer (2009) The Journal of biological chemistry 284(14), 9465-9474) driving the expression of recombinant proteins underthe control of the T7 promoter. PCR products digested with BglII werecloned into the BamHI site of pET22b-Strep.

TABLE 4 Primers used in this study.Restriction sites are highlighted in bold. Primer pairResulting construct GC GGATCC AAAGTCTTAACC StrepII-CsaA-His₆ GTCTTTGGCCCG CTCGAG TCTATTCTTTA ATAAAGTTTCTACA GC AGATCT TTTATACTTAATStrepII-CsaB-His₆ AACAGAAAATGGC CCG CTCGAG TTTCTCAAATG ATGATGGTAATG CCGCTCGAG TTTCTCAAATG StrepII-Δ69-CsaB-His₆ ATGATGGTAATG GC AGATCTATGTTAATTCCT ATTAATTTTTTTAA CCG CTCGAG TTTCTCAAATG Δ69-CsaB-His₆ATGATGGTAATG GCATCT CATATG TTAATTCC TATTAATTTTTTTTAATTT GCATCT CATATGCTGATCCC Δ69-CsaB_(Co)-His₆ GATCAATTTCTTT CCG CTCGAG TTTCTCGAAGGAGCTCGGC CCG CTCGAG TATATTTTGGA StrepII-CsaC-His₆ TTATGGT GC GGATCCTTATCTAATTTA AAAACAGG

Expression and Purification of Recombinant CsaA, CsaB and CsaC—

Freshly transformed E. coli BL21(DE3) were grown at 15° C. in PowerBrothmedium for 18 h. At an optical density of OD₆₀₀=1.0 protein expressionwas induced by addition of 0.1 mM IPTG and allowed to proceed for aperiod of 20 h. In test expressions, 0.2 ml of culture-volume werepelleted with 16,000×g for 1 min. Cell pellets were lysed with 0.1 mllysis buffer (50 mM Tris pH 8.0, 2 mM EDTA, 0.1 mg/ml lysozyme). Thelysis was intensified by 3 cycles of sonication (Branson sonifier 450,100% amplitude) interrupted by 3 min of cooling on ice. Soluble andinsoluble fractions were separated by centrifugation (16,000×g, 30 min,4° C.), the supernatant mixed (1:1) with Laemmli-buffer and used forPAGE as described below.

For protein purification, pellets from 125 mL expression culture werepelleted by centrifugation (6,000×g, 10 min, 4° C.). After a washingstep with PBS, cells were re-suspended in 7.5 ml binding buffer (50 mMTris pH 8.0, 300 mM NaCl) complemented with 40 μg/ml Bestatin (Sigma), 1μg/ml Pepstatin (Applichem), 100 μM PMSF (Stratagene) and sonified(Branson Digital Sonifier, 50% amplitude, 8×30 s, interrupted by coolingon ice). After centrifugation at 27,000×g for 30 min, the solublefractions were directly loaded onto a HisTrap columns (GE Healthcare) toenrich the recombinant proteins by immobilized metal ion affinitychromatography (IMAC). Columns were washed with binding buffer (50 mMTris pH 8.0, 300 mM NaCl) and proteins eluted in step gradients using10%, 30%, 50% and 100% elution buffer (binding buffer containing 500 mMimidazol). Fractions containing recombinant protein were pooled and thebuffer exchanged to storage buffer (50 mM Tris pH 8.0, 50 mM NaCl forCsaA/CsaB; 50 mM Hepes pH 7.05, 100 mM NaCl, 5 mM MgCl2 and 1 mM EDTAfor CsaC) using the HiPrep 26/10 Desalting column (GE Healthcare).Isolated proteins were concentrated using Amicon Ultra centrifugaldevices (Millipore 30 MWCO). After separation into aliquots, sampleswere snap frozen in liquid nitrogen and stored at −80° C.

SDS-PAGE and Immunoblotting—

SDS-PAGE was performed under reducing conditions using 2.5% (v/v)1-mercaptoethanol and 1.5% (w/v) SDS. Proteins were stained usingRoti-Blue (Carl Roth GmbH) according to the manufacturer's guidelines.For western blot analysis samples and standard proteins were blottedonto PVDF membranes (Millipore). His-tagged proteins were detected with0.5 μg/ml anti-penta-His antibody (Qiagen) and goat anti-mouse IR680 orgoat anti-mouse IR800 antibody (LI-COR) as second antibody. Secondantibodies were used in a 1:20,000 dilution.

Preparation of CPSA Oligosaccharides—

CPSA oligosaccharide samples with an averaged degree of polymerization(avDP) of 6 and 15, respectively, were generated by acidic hydrolysis oflong CPSA chains isolated from bacterial cultures (CPSA_(n)). Therefore,solutions containing 2.5 mg CPSA/mL sodium acetate buffer (50 mM sodiumacetate, pH 4.8) were incubated at 73° C. for 6 h and two pool fractions(avDP 6 and 15, respectively) were purified by anionic exchangechromatography (Q-Sepharose column, GE Healthcare) using a sodiumacetate gradient. The avDP and the dispersion of saccharide chains wasdetermined by ³¹P NMR and High Performance Anionic ExchangeChromatography-Pulsed Amperometric Detection (HPAEC-PAD) analysisfollowing an established protocol (Berti (2012) Vaccine 30 (45),6409-6415). If used in enzymatic reactions, hydrolyzed CPSA (CPSA_(hyd))was dephosphorylated (CPSA_(hyd)-deP) using acid phosphatase (Sigma)according to the manufacturer's guidelines.

Activity Testing of CsaA/CsaB by Use of a Radioactive Assay System—

CsxA/CsaB activity was analysed using an adaptation of a radioactiveincorporation assay previously described for theN-acetylglucosamine-1-phosphate-transferase from NmX (Fiebig,Glycobiology (2014) 24:150-8). Briefly, assays were carried out with 5μl of the soluble fractions of bacterial lysates expressing eitherrecombinant CsaB or CsaA (see FIG. 22C) or with purified andepitope-tagged proteins (112 pmol StrepII-CsaA-His₆; 88 pmolΔ69CsaB_(co)-His₆) in a total volume of 25 μl assay buffer (50 mM TrispH 8.0 or various pH for determination of the pH optimum). Divalentcations were added from stock solutions. The reaction was primed with 5ng avDP15 and started by the addition of 0.05 μmol UDP-GlcNAc(Calbiochem) containing 0.05 ρCi UDP-[¹⁴C]-GlcNAc (American RadiolabeledChemicals). Samples were incubated at 37° C. and 5 μl aliquots spottedonto Whatman 3MM CHR paper after 0, 5, 10 and 30 min. Followingdescending paper chromatography, the chromatographically immobile¹⁴C-labeled CPSA was quantified by scintillation counting.

Activity Testing of CsaA/CsaB by Use of a Multi-EnzymeSpectrophotometric Assay—

1.2 μM of CsaA and 1 μM CsaB were assayed in the presence of 0.25 mMUDP-GlcNAc (Calbiochem), 20 mM MgCl₂ and 50 mM Tris pH 8.0 in a totalvolume of 100 μL. The consumption of UDP-GlcNAc was coupled tonicotinamide adenine dinucleotide (NADH) consumption using the followingenzymes/substrates: 0.25 mM adenosine triphosphate (ATP, Roche), 1 mMphosphoenolpyruvate (PEP, ABCR), 0.3 mM (NADH, Roche), 9-15 units/mlpyruvate kinase, 13.5-21 units/ml lactic dehydrogenase (PK/LDH mixSigma), 0.05 mg/ml nucleoside monophosphate kinase (Roche). Absorptionwas measured at 340 nm every 30 min using a Biotek EL 808 96-well platereader.

Physicochemical Analysis of CPSA_(iv).

To produce sufficient CPSA (CPSA_(iv)) for PAGE and NMR analyses, 0.84nmol (1.2 μM final) of StrepII-CsaA-His₆ and 37.5 pmol (50 nM final) ofΔ69CsaB_(co)-His₆ in reaction buffer (50 mM Tris pH 8.0, 20 mM MgCl₂)were incubated with 5 mM UDP-GlcNAc and 6.8 μg of CPSA_(hyd) of avDP6after de-O-acetylation and dephosphorylation (CPSA_(hyd(deOAc))-deP).The total reaction volume was adjusted to 750 μl. In control 1 alsoStrepII-CsaA-His₆ was used at 50 nM. Reactions as well as controlsamples were incubated over night at 37° C. 5 μl of each sample werethen used for separation on high percentage (25%) PAGE and visualized bya combined Alcian blue/silver staining procedure (Min (1986) Analyticalbiochemistry 155 (2), 275-285).

The residual sample was freeze-dried, solubilized in 0.75 ml deuteriumoxide (D₂O, 99.9% atom D; Aldrich) to give a concentration of 0.5-1 mgsaccharide and used for product characterization by NMR. All the ¹H and³¹P NMR experiments were recorded as previously described (Fiebig,Glycobiology. (2014) 24:150-8).

HPLC-AEC was performed on a Prominence UFLC-XR (Shimadzu) equipped witha CarboPac PA-100 column (2×250 mm, Dionex). Samples were separated asdescribed by (Keys (2012) Analytical biochemistry 427 (2), 107-115) withthe minor adjustment that H₂O and 1M NaCl were used as mobile phases M₁and M₂, respectively. 5 μl of the samples were loaded for the detectionof nucleotides at 280 nm and 50 μl for the detection of CPSA at 214 nm.Products were separated using an elution gradient consisting of a −2curved gradient from 0 to 30% M₂ over 4 min followed by a lineargradient from 30 to 84% M₂ over 33 min. Enzyme concentrations were usedas indicated in FIG. 6. All other reactants were used in the amountsdescribed above.

Analysis of 2-acetamidoglucal—

Assignments of 1H NMR spectrum were in agreement with those reported inliterature (29). ¹H NMR (D₂O, 400 MHz): δ=6.68 (d, 1H, J_(1,2) 1.0,H-1), 4.25 (dd, H 1, J_(3,4) 6.5 Hz, H-3), 3.99 (dt, 1H, J_(4,5) 8.4,J_(5,6a)=J_(5,6b) 4.2 Hz, 6.5 Hz, H-5), 3.86 (d, 2H, H-6), 3.77 (dd, 1H,H-4), 2.05 (s, 3H, CH₃CO). Significant signals from ¹³C NMR (D₂O, 100MHz): δ=141.47 (C-1), 78.70 (C-5), 68.70 (C-3), 68.36 (C-4), 59.84(C-6), 21.84 (2×CH₃CO).

In Vitro Synthesis, Purification and Immunological Analysis of CPSA_(iv)and CPSA_(iv(OAc))—

To generate CPSA_(iv), 10 nmol CsaA and 16 nmol CsaB were incubated overnight at 37° C. in reaction buffer (50 mM Tris pH 8.0, 20 mM MgCl₂)complemented with 10 mM UDP-GlcNAc in a total volume of 9 ml. Thereaction was primed with 1 μg of CPSA_(hyd(deOAc))-deP of avDP6.Acetylation of 1 mg CPSA_(iv) was performed for 4 h at 37° C. in thepresence of 1.2 nmol CsaC and 14 mM acetyl-CoA (Sigma) in a total volumeof 0.5 ml acetylation buffer (25 mM Tris pH 7.5, 50 mM NaCl). BothCPSA_(iv) and CPSA_(iv(OAc)) were purified via anion exchangechromatography (AEC) using a MonoQ HR5/5 column (Pharmacia biotech) at aflow rate of 1 ml/min and a linear sodium chloride gradient. CPScontaining fractions eluting at 540 mM NaCl were pooled, dialysed(ZelluTrans, Roth, 1 kDa MWCO) against water and freeze dried forfurther analysis. For dot blot analyses, small aliquots of the purifiedCPSA_(iv) and CPSA_(iv(OAc)) was spotted onto nitro-cellulose (Whatman)and incubated with mAb 932 specifically directed against CPSA_((OAc))(mAb 935 was generated in the laboratory of Prof. Dr. D.Bitter-Suermann, Hannover Medical School, Institute for MedicalMicrobiology, and was kindly provided for this study) in a 1:10,000dilution. Dot blots were developed with goat anti-mouse IR800 antibody(LI-COR) in 1:20,000 dilution.

EXAMPLE 5 Design and Expression of N- and C-Terminally TruncatedCsxA-Constructs Carrying an N-Terminal MBP- and a C-Terminal His₆-Tag

The materials and methods for this Example are described herein aboveand also shown in Fiebig, Glycobiology (2014) 24:150-8.

The secondary structure prediction software PHYRE (Kelley et al., 2009,Nat Protoc. 4, 363-371) was used to predict secondary structure elementswithin the CsxA amino acid sequence. Amplification of csxA using thetruncation primers FF52 and FF53, annealing upstream of the conservedregion (CR) 1 and downstream of CR2 in unstructured regions where nosecondary structure was predicted, resulted in a dN58- or dC99-truncatedCsxA, respectively.

The PCR products were cloned into a modified pET43.1 vector enabling theexpression of the truncated genes as N-terminally MBP- and C-terminallyHis₆-tagged fusion constructs. After expression in BL21(DE3)Goldfollowed by lysis of the pelletized cells, soluble fractions were usedin a radioactive incorporation assay. Following SDS-PAGE and subsequentimmuno-blotting against the His₆-tag, the Odyssey-Software could be usedto determine the amount of the respective constructs within the solubleand the insoluble fraction. The former was used to normalize theenzymatic activity obtained in the radioactive incorporation assay.

Judging from the western blot (FIG. 30. B), both full-length andtruncated CsxA were expressed in good yields. The truncation of theN-terminus increased the ratio of soluble:insoluble construct from 2.4(full-length) to 4.4, while the truncation of the C-terminus decreasedthe ratio to 1.2. However, the absolute amount of the ΔC99 truncationwithin the soluble fraction was comparable to the amount of full-lengthconstruct. The amount of ΔN58CsxA was increased by 20%. Of note, theradioactive incorporation assay showed that the activity of both theN-terminal and the C-terminal truncation was increased by 38% and 61%,respectively, if compared to the activity of the full-length construct.

With the aim of analyzing the influence of the termini towards thesolubility of CsxA and furthermore defining the minimal catalytic domainof the enzyme, the truncated constructs shown in FIG. 29 were generated.It could be shown that the truncation of the N-terminus by 58 aaresulted in an increase of the ratio of the amount of protein betweensoluble and insoluble fraction by 80%. Moreover, the enzymatic activitywas increased by 38% if compared to the full-length construct.

Group II polymerases are supposed to be part of the capsule transportcomplex (Steenbergen (2008) Mol. Microbiol. 68, 1252-1267). The improvedsolubility could indicate that hydrophobic aa within the first 58 aa ofthe CsxA sequence are exposed on the enzyme's surface to undergoprotein-protein interactions with other components of the capsuletransport complex.

The truncation of the C-terminus led to an increase of activity by 61%(compared to full-length). However, solubility was reduced by 50%. Sincethe C-terminus might play a role in the formation of the correctoligomerisation state of CsxA, the loss of the correct oligomerisationof ΔC99CsxA might explain the reduced solubility. However, furtherinvestigation is needed to understand these structure-functionrelationships.

The truncation studies showed that the minimal catalytic domain of CsxAcan be reduced to a polypeptide of 329 aa, which corresponds to areduction of 33% of the full-length sequence. Furthermore, the resultsindicate that the truncated termini do not contain aa of CsxA's activecenter.

Further Studies

In later studies, the ΔN58ΔC99 double truncation was generated andcloned, as well as all afore mentioned construct, into optimisedexpression vectors. The optimised expression vector is described inFiebig (Glycobiology (2014) 24:150-8) as MBP-CsxA-His₆ (tac).

In accordance with the results described above, the truncation of theN-terminus led to an increase in purification yield by more than 500%,whereas the purification yield of the C-terminal truncation wascomparable to the full-length construct (FIG. 31C).

Activity of the purified constructs, measured using an adaption of themulti-enzyme assay described by Freiberger et al. (2007, MolecularMicrobiology 65, 1258-1275), confirmed the increased activity of bothsingle CsxA truncations (FIG. 31D). However, the activity of the doubletruncation was comparable to the activity measured for the full-lengthconstruct.

Analytical size-exclusion chromatography showed that the C-terminus isnot involved in oligomerisation/aggregation, since the C-terminaltruncation, as well as the full-length enzyme (see Fiebig, Glycobiology.(2014) 24:150-8) forms aggregates in low salt buffer (FIG. 31A) and tri-to tetrameric assemblies in high salt buffer (FIG. 31B).

EXAMPLE 6 Regulation of ΔC99-CP-X and ΔN58ΔC99-CP-X by the Reaction Time

The materials and methods for this Example are described herein aboveand also shown in Fiebig, Glycobiology (2014) 24:150-8.

The C-terminally (e.g. ΔC99-CP-X) as well as the C- and N-terminally(e.g. ΔN58ΔC99-CP-X) truncated version of CP-X show a distributivepolymerisation (FIG. 32). In addition, for both polypeptides the lengthof the produced capsular polysaccharides can be regulated via thereaction time (FIG. 32). However, if the C- and N-terminally truncatedCP-X is regulated by the reaction time, the polydispersity is lower asit the C-terminally truncated version is used. Therefore, the C- andN-terminally truncated CP-X is the polypeptide which is most suitablefor a regulation via the reaction time.

EXAMPLE 7 Production of Capsular Polysaccharides Using a CapsulePolymerase Coupled to a Solid Phase

Preparation of the Acceptor Substrate and Determination of theDonor:Acceptor Ratio (d/a).

Oligosaccharide fractions were prepared as described in Example 1 andfractions containing DP2-10 were pooled to get a mixture of shortoligosaccharides ranging from DP2-10. Using the ΔN58ΔC99 CsxAtruncation, the donor to acceptor ratio was experimentally determinedusing 10 mM UDP-GlcNAc and variable amounts of acceptor substrate inover-night reactions to give a product distribution ranging fromDP10-DP60 (FIG. 34 a). All obtained product pools were within theDP10-DP60 range and the lowest d/a was chosen for the subsequentexperiment. In particular, the d/a was approximately between 100:1 and200:1.

Expression of the Enzyme and Coupling of the Construct to the Column

ΔN58ΔC99 CsxA was expressed in a 50 mL culture volume and purified viaHis-Trap as described in Fiebig et al 2014a. Briefly, the cells werelysed by sonication in binding buffer (50 mM Tris pH 8.0, 1 mM DTT, 500mM NaCl) and coupled to a 1 mL His-Trap column. The column was washedwith binding buffer containing 37.5 mM imidazole to remove cell debrisand non-specifically bound protein before it was equilibrated with abuffer only containing 50 mM Tris pH 8.0 and 1 mM DTT.

Production of CPSX Using ΔN58ΔC99 CsxA Coupled to a Solid Phase

The reaction mix containing 50 mM Tris pH 8.0, 10 mM UDP-GlcNAc, 20 mMMgCl2, 1 mM DTT and the amount of acceptor determined in FIG. 34A wasup-scaled to 5 mL and loaded onto the His-Trap column containing theimmobilized ΔN58ΔC99 CsxA. The mix was circulated through the columnusing a Peristaltic P1 pump (GE Healthcare) for 2 h and aliquots weretaken every 20 min. Aliquots were snap-frozen in liquid nitrogen andΔN58ΔC99 CsxA was subsequently inactivated for 2.5 min at 98° C. Theanalysis was performed using the HPLC-assay described in Example 4.

Following the consumption of the donor substrate UDP-GlcNAc and theproduction of the second reaction product UMP at 280 nm revealed thatthe reaction was finished after 20 min and shows that ΔN58ΔC99 CsxA isactive even if it is bound/coupled to a solid phase (His-Trap beads)(FIG. 34B). Moreover, the HPLC-AEC (214 nm) analysis of the synthesizedCPSX revealed that the ability of the distributive ΔN58ΔC99 CsxA tosynthesize narrow product distribution is maintained even if theconstruct is coupled to a solid phase. The enzymatically produced CPSXpool is well within the range of the avDP15 pool (i.e. between DP10 andDP60) which is commonly used for vaccine manufacture (FIG. 24C).ΔN58ΔC99 CsxA was eluted after the reaction had been finished todemonstrate that it remained coupled to the column during the period ofthe reaction (FIG. 34D). The above presented data demonstrates that CPSXproduction using CsxA can be performed on a solid phase, thus omittingfurther purification steps to remove the enzyme from the CPSX fraction.

The present invention refers to the following nucleotide and amino acidsequences:

SEQ ID NO: 1: DNA CP-A/CsaB wildtyp/full-length (1632)atgtttatacttaataacagaaaatggcgtaaacttaaaagagaccctagcgctttctttcgagatagtaaatttaactttttaagatatttttctgctaaaaaatttgcaaagaattttaaaaattcatcacatatccataaaactaatataagtaaagctcaatcaaatatttcttcaaccttaaaacaaaatcggaaacaagatatgttaattcctattaatttttttaattttgaatatatagttaaaaaacttaacaatcaaaacgcaataggtgtatatattcttccttctaatcttactcttaagcctgcattatgtattctagaatcacataaagaagactttttaaataaatttcttcttactatttcctctgaaaatttaaagcttcaatacaaatttaatggacaaataaaaaatcctaagtccgtaaatgaaatttggacagatttatttagcattgctcatgttgacatgaaactcagcacagatagaactttaagttcatctatatctcaattttggttcagattagagttctgtaaagaagataaggattttatcttatttcctacagctaacagatattctagaaaactttggaagcactctattaaaaataatcaattatttaaagaaggcatacgaaactattcagaaatatcttcattaccctatgaagaagatcataattttgatattgatttagtatttacttgggtcaactcagaagataagaattggcaagagttatataaaaaatataagcccgactttaatagcgatgcaaccagtacatcaagattccttagtagagatgaattaaaattcgcattacgctcttgggaaatgaatggatccttcattcgaaaaatttttattgtctctaattgtgctcccccagcatggctagatttaaataaccctaaaattcaatgggtatatcacgaagaaattatgccacaaagtgcccttcctacttttagctcacatgctattgaaaccagcttgcaccatataccaggaattagtaactattttatttacagcaatgacgacttcctattaactaaaccattgaataaagacaatttcttctattcgaatggtattgcaaagttaagattagaagcatggggaaatgttaatggtgaatgtactgaaggagaacctgactacttaaatggtgctcgcaatgcgaacactctcttagaaaaggaatttaaaaaatttactactaaactacatactcactcccctcaatccatgagaactgatattttatttgagatggaaaaaaaatatccagaagagtttaatagaacactacataataaattccgatctttagatgatattgcagtaacgggctatctctatcatcattatgccctactctctggacgagcactacaaagttctgacaagacggaacttgtacagcaaaatcatgatttcaaaaagaaactaaataatgtagtgaccttaactaaagaaaggaattttgacaaacttcctttgagcgtatgtatcaacgatggtgctgatagtcacttgaatgaagaatggaatgttcaagttattaagttcttagaaactcttttcccattaccatcatcatttgag aaa SEQ ID NO: 2:DNA CP-A/CsaB codon optimized (without)ATGTTCATCTTGAACAACCGCAAATGGCGCAAATTGAAGCGTGACCCAAGCGCGTTTTTTCGTGACAGCAAATTCAACTTTCTGCGCTATTTCTCCGCGAAAAAGTTTGCGAAGAATTTCAAAAACAGCTCGCATATCCATAAAACCAACATTAGCAAAGCGCAGTCCAATATTTCCAGCACCTTGAAGCAG AACCGTAAGCAGGAT ATGCTGATCCCGATCAATTTCTTTAATTTTGAGTACATCGTGAAGAAACTGAATAACCAAAACGCAATCGGCGTGTACATTCTGCCGTCTAATCTGACCCTGAAACCAGCATTGTGCATCTTGGAGTCGCACAAAGAGGACTTCCTGAACAAATTTTTGTTGACCATTAGCAGCGAGAACCTGAAACTGCAGTATAAGTTCAATGGTCAGATCAAAAATCCGAAAAGCGTGAACGAAATCTGGACCGACCTGTTTAGCATTGCTCACGTCGACATGAAGCTGAGCACCGACCGTACGCTGTCCTCGTCCATCAGCCAATTTTGGTTTCGCCTGGAGTTCTGTAAAGAGGACAAGGACTTCATCCTGTTTCCGACGGCAAATCGTTACAGCCGCAAGCTGTGGAAGCACAGCATCAAAAATAATCAGCTGTTTAAGGAAGGTATCCGTAACTACAGCGAGATTAGCTCGCTGCCGTACGAGGAAGACCATAACTTCGACATCGATCTGGTCTTTACCTGGGTCAATTCGGAAGACAAAAACTGGCAGGAACTGTACAAGAAATATAAGCCGGATTTTAATAGCGATGCCACCTCGACGAGCCGTTTTCTGAGCCGTGACGAGCTGAAGTTTGCGCTGCGCTCGTGGGAAATGAACGGTAGCTTCATCCGTAAAATCTTTATCGTCAGCAACTGCGCGCCGCCGGCCTGGCTGGATCTGAACAATCCGAAGATCCAATGGGTGTATCACGAGGAGATCATGCCACAGAGCGCCCTGCCAACCTTCAGCAGCCATGCTATTGAGACTAGCTTGCATCACATTCCGGGCATCTCCAACTACTTCATCTACTCTAATGACGATTTTCTGTTGACCAAACCGCTGAACAAAGACAACTTCTTTTACTCCAACGGTATTGCTAAACTGCGTCTGGAAGCCTGGGGTAACGTTAACGGTGAATGTACCGAAGGCGAGCCGGATTACCTGAACGGCGCGCGTAACGCAAATACGCTGCTGGAGAAAGAGTTTAAAAAGTTTACCACCAAGCTGCACACCCACAGCCCGCAGAGCATGCGTACCGACATCCTGTTCGAGATGGAGAAAAAATACCCAGAAGAGTTCAATCGCACGCTGCACAACAAGTTCCGCAGCCTGGATGACATCGCGGTTACCGGCTACCTGTACCATCACTACGCATTGCTGTCTGGCCGCGCTCTGCAATCCAGCGATAAGACCGAACTGGTCCAGCAGAATCACGACTTTAAAAAGAAGCTGAATAATGTTGTCACCCTGACCAAAGAGCGTAACTTTGATAAGCTGCCGCTGAGCGTTTGTATTAATGACGGTGCAGACAGCCACCTGAATGAGGAGTGGAATGTGCAAGTTATCAAATTCTTGGAGACCTTGTTCCCGTTGCCGAGCTCCTTCGAG AAA SEQ ID NO: 3:DNA ΔN69-CP-A (codon optimized) (1425)CTGATCCCGATCAATTTCTTTAATTTTGAGTACATCGTGAAGAAACTGAATAACCAAAACGCAATCGGCGTGTACATTCTGCCGTCTAATCTGACCCTGAAACCAGCATTGTGCATCTTGGAGTCGCACAAAGAGGACTTCCTGAACAAATTTTTGTTGACCATTAGCAGCGAGAACCTGAAACTGCAGTATAAGTTCAATGGTCAGATCAAAAATCCGAAAAGCGTGAACGAAATCTGGACCGACCTGTTTAGCATTGCTCACGTCGACATGAAGCTGAGCACCGACCGTACGCTGTCCTCGTCCATCAGCCAATTTTGGTTTCGCCTGGAGTTCTGTAAAGAGGACAAGGACTTCATCCTGTTTCCGACGGCAAATCGTTACAGCCGCAAGCTGTGGAAGCACAGCATCAAAAATAATCAGCTGTTTAAGGAAGGTATCCGTAACTACAGCGAGATTAGCTCGCTGCCGTACGAGGAAGACCATAACTTCGACATCGATCTGGTCTTTACCTGGGTCAATTCGGAAGACAAAAACTGGCAGGAACTGTACAAGAAATATAAGCCGGATTTTAATAGCGATGCCACCTCGACGAGCCGTTTTCTGAGCCGTGACGAGCTGAAGTTTGCGCTGCGCTCGTGGGAAATGAACGGTAGCTTCATCCGTAAAATCTTTATCGTCAGCAACTGCGCGCCGCCGGCCTGGCTGGATCTGAACAATCCGAAGATCCAATGGGTGTATCACGAGGAGATCATGCCACAGAGCGCCCTGCCAACCTTCAGCAGCCATGCTATTGAGACTAGCTTGCATCACATTCCGGGCATCTCCAACTACTTCATCTACTCTAATGACGATTTTCTGTTGACCAAACCGCTGAACAAAGACAACTTCTTTTACTCCAACGGTATTGCTAAACTGCGTCTGGAAGCCTGGGGTAACGTTAACGGTGAATGTACCGAAGGCGAGCCGGATTACCTGAACGGCGCGCGTAACGCAAATACGCTGCTGGAGAAAGAGTTTAAAAAGTTTACCACCAAGCTGCACACCCACAGCCCGCAGAGCATGCGTACCGACATCCTGTTCGAGATGGAGAAAAAATACCCAGAAGAGTTCAATCGCACGCTGCACAACAAGTTCCGCAGCCTGGATGACATCGCGGTTACCGGCTACCTGTACCATCACTACGCATTGCTGTCTGGCCGCGCTCTGCAATCCAGCGATAAGACCGAACTGGTCCAGCAGAATCACGACTTTAAAAAGAAGCTGAATAATGTTGTCACCCTGACCAAAGAGCGTAACTTTGATAAGCTGCCGCTGAGCGTTTGTATTAATGACGGTGCAGACAGCCACCTGAATGAGGAGTGGAATGTGCAAGTTATCAAATTCTTGGAGACCTTGTTCCCGTTGCCGAGCTCCTTCGAGAAA SEQ ID NO: 4: DNA ΔN97-CP-A (1344)ccttctaatcttactcttaagcctgcattatgtattctagaatcacataaagaagactttttaaataaatttcttcttactatttcctctgaaaatttaaagcttcaatacaaatttaatggacaaataaaaaatcctaagtccgtaaatgaaatttggacagatttatttagcattgctcatgttgacatgaaactcagcacagatagaactttaagttcatctatatctcaattttggttcagattagagttctgtaaagaagataaggattttatcttatttcctacagctaacagatattctagaaaactttggaagcactctattaaaaataatcaattatttaaagaaggcatacgaaactattcagaaatatcttcattaccctatgaagaagatcataattttgatattgatttagtatttacttgggtcaactcagaagataagaattggcaagagttatataaaaaatataagcccgactttaatagcgatgcaaccagtacatcaagattccttagtagagatgaattaaaattcgcattacgctcttgggaaatgaatggatccttcattcgaaaaatttttattgtctctaattgtgctcccccagcatggctagatttaaataaccctaaaattcaatgggtatatcacgaagaaattatgccacaaagtgcccttcctacttttagctcacatgctattgaaaccagcttgcaccatataccaggaattagtaactattttatttacagcaatgacgacttcctattaactaaaccattgaataaagacaatttcttctattcgaatggtattgcaaagttaagattagaagcatggggaaatgttaatggtgaatgtactgaaggagaacctgactacttaaatggtgctcgcaatgcgaacactctcttagaaaaggaatttaaaaaatttactactaaactacatactcactcccctcaatccatgagaactgatattttatttgagatggaaaaaaaatatccagaagagtttaatagaacactacataataaattccgatctttagatgatattgcagtaacgggctatctctatcatcattatgccctactctctggacgagcactacaaagttctgacaagacggaacttgtacagcaaaatcatgatttcaaaaagaaactaaataatgtagtgaccttaactaaagaaaggaattttgacaaacttcctttgagcgtatgtatcaacgatggtgctgatagtcacttgaatgaagaatggaatgttcaagttattaagttcttagaaactcttttcccattaccatcatcatttgagaaa SEQ ID NO: 5:DNA ΔN167-CP-A (1134) Actttaagttcatctatatctcaattttggttcagattagagttctgtaaagaagataaggattttatcttatttcctacagctaacagatattctagaaaactttggaagcactctattaaaaataatcaattatttaaagaaggcatacgaaactattcagaaatatcttcattaccctatgaagaagatcataattttgatattgatttagtatttacttgggtcaactcagaagataagaattggcaagagttatataaaaaatataagcccgactttaatagcgatgcaaccagtacatcaagattccttagtagagatgaattaaaattcgcattacgctcttgggaaatgaatggatccttcattcgaaaaatttttattgtctctaattgtgctcccccagcatggctagatttaaataaccctaaaattcaatgggtatatcacgaagaaattatgccacaaagtgcccttcctacttttagctcacatgctattgaaaccagcttgcaccatataccaggaattagtaactattttatttacagcaatgacgacttcctattaactaaaccattgaataaagacaatttcttctattcgaatggtattgcaaagttaagattagaagcatggggaaatgttaatggtgaatgtactgaaggagaacctgactacttaaatggtgctcgcaatgcgaacactctcttagaaaaggaatttaaaaaatttactactaaactacatactcactcccctcaatccatgagaactgatattttatttgagatggaaaaaaaatatccagaagagtttaatagaacactacataataaattccgatctttagatgatattgcagtaacgggctatctctatcatcattatgccctactctctggacgagcactacaaagttctgacaagacggaacttgtacagcaaaatcatgatttcaaaaagaaactaaataatgtagtgaccttaactaaagaaaggaattttgacaaacttcctttgagcgtatgtatcaacgatggtgctgatagtcacttgaatgaagaatggaatgttcaagttattaagttcttagaaactcttttcccattaccatcatcatttgagaaa SEQ ID NO: 6: DNA ΔN235-CP-A (933)gatattgatttagtatttacttgggtcaactcagaagataagaattggcaagagttatataaaaaatataagcccgactttaatagcgatgcaaccagtacatcaagattccttagtagagatgaattaaaattcgcattacgctcttgggaaatgaatggatccttcattcgaaaaatttttattgtctctaattgtgctcccccagcatggctagatttaaataaccctaaaattcaatgggtatatcacgaagaaattatgccacaaagtgcccttcctacttttagctcacatgctattgaaaccagcttgcaccatataccaggaattagtaactattttatttacagcaatgacgacttcctattaactaaaccattgaataaagacaatttcttctattcgaatggtattgcaaagttaagattagaagcatggggaaatgttaatggtgaatgtactgaaggagaacctgactacttaaatggtgctcgcaatgcgaacactctcttagaaaaggaatttaaaaaatttactactaaactacatactcactcccctcaatccatgagaactgatattttatttgagatggaaaaaaaatatccagaagagtttaatagaacactacataataaattccgatctttagatgatattgcagtaacgggctatctctatcatcattatgccctactctctggacgagcactacaaagttctgacaagacggaacttgtacagcaaaatcatgatttcaaaaagaaactaaataatgtagtgaccttaactaaagaaaggaattttgacaaacttcctttgagcgtatgtatcaacgatggtgctgatagtcacttgaatgaagaatggaatgttcaagttattaagttcttagaaactcttttccca ttaccatcatcatttgagaaaSEQ ID NO: 7: DNA ΔC45-CP-A (1497)tttatacttaataacagaaaatggcgtaaacttaaaagagaccctagcgctttctttcgagatagtaaatttaactttttaagatatttttctgctaaaaaatttgcaaagaattttaaaaattcatcacatatccataaaactaatataagtaaagctcaatcaaatatttcttcaaccttaaaacaaaatcggaaacaagatatgttaattcctattaatttttttaattttgaatatatagttaaaaaacttaacaatcaaaacgcaataggtgtatatattcttccttctaatcttactcttaagcctgcattatgtattctagaatcacataaagaagactttttaaataaatttcttcttactatttcctctgaaaatttaaagcttcaatacaaatttaatggacaaataaaaaatcctaagtccgtaaatgaaatttggacagatttatttagcattgctcatgttgacatgaaactcagcacagatagaactttaagttcatctatatctcaattttggttcagattagagttctgtaaagaagataaggattttatcttatttcctacagctaacagatattctagaaaactttggaagcactctattaaaaataatcaattatttaaagaaggcatacgaaactattcagaaatatcttcattaccctatgaagaagatcataattttgatattgatttagtatttacttgggtcaactcagaagataagaattggcaagagttatataaaaaatataagcccgactttaatagcgatgcaaccagtacatcaagattccttagtagagatgaattaaaattcgcattacgctcttgggaaatgaatggatccttcattcgaaaaatttttattgtctctaattgtgctcccccagcatggctagatttaaataaccctaaaattcaatgggtatatcacgaagaaattatgccacaaagtgcccttcctacttttagctcacatgctattgaaaccagcttgcaccatataccaggaattagtaactattttatttacagcaatgacgacttcctattaactaaaccattgaataaagacaatttcttctattcgaatggtattgcaaagttaagattagaagcatggggaaatgttaatggtgaatgtactgaaggagaacctgactacttaaatggtgctcgcaatgcgaacactctcttagaaaaggaatttaaaaaatttactactaaactacatactcactcccctcaatccatgagaactgatattttatttgagatggaaaaaaaatatccagaagagtttaatagaacactacataataaattccgatctttagatgatattgcagtaacgggctatctctatcatcattatgccctactctctggacgagcactacaaagttctgacaagacggaacttgtacagcaaaatcatgatttcaaaaagaaactaaataatgtagtgacc ttaactaaa SEQ ID NO: 8:DNA ΔC25-CP-A (1557) tttatacttaataacagaaaatggcgtaaacttaaaagagaccctagcgctttctttcgagatagtaaatttaactttttaagatatttttctgctaaaaaatttgcaaagaattttaaaaattcatcacatatccataaaactaatataagtaaagctcaatcaaatatttcttcaaccttaaaacaaaatcggaaacaagatatgttaattcctattaatttttttaattttgaatatatagttaaaaaacttaacaatcaaaacgcaataggtgtatatattcttccttctaatcttactcttaagcctgcattatgtattctagaatcacataaagaagactttttaaataaatttcttcttactatttcctctgaaaatttaaagcttcaatacaaatttaatggacaaataaaaaatcctaagtccgtaaatgaaatttggacagatttatttagcattgctcatgttgacatgaaactcagcacagatagaactttaagttcatctatatctcaattttggttcagattagagttctgtaaagaagataaggattttatcttatttcctacagctaacagatattctagaaaactttggaagcactctattaaaaataatcaattatttaaagaaggcatacgaaactattcagaaatatcttcattaccctatgaagaagatcataattttgatattgatttagtatttacttgggtcaactcagaagataagaattggcaagagttatataaaaaatataagcccgactttaatagcgatgcaaccagtacatcaagattccttagtagagatgaattaaaattcgcattacgctcttgggaaatgaatggatccttcattcgaaaaatttttattgtctctaattgtgctcccccagcatggctagatttaaataaccctaaaattcaatgggtatatcacgaagaaattatgccacaaagtgcccttcctacttttagctcacatgctattgaaaccagcttgcaccatataccaggaattagtaactattttatttacagcaatgacgacttcctattaactaaaccattgaataaagacaatttcttctattcgaatggtattgcaaagttaagattagaagcatggggaaatgttaatggtgaatgtactgaaggagaacctgactacttaaatggtgctcgcaatgcgaacactctcttagaaaaggaatttaaaaaatttactactaaactacatactcactcccctcaatccatgagaactgatattttatttgagatggaaaaaaaatatccagaagagtttaatagaacactacataataaattccgatctttagatgatattgcagtaacgggctatctctatcatcattatgccctactctctggacgagcactacaaagttctgacaagacggaacttgtacagcaaaatcatgatttcaaaaagaaactaaataatgtagtgaccttaactaaagaaaggaattttgacaaacttcctttgagcgtatgtatc aacgatggtgctgatagtcacSEQ ID NO: 9: AS CP-A/CsaB wildtyp/full-length (544)MFILNNRKWRKLKRDPSAFFRDSKFNFLRYFSAKKFAKNFKNSSHIHK TNISKAQSNISSTLKQNRKQD MLIPINFFNFEYIVKKLNNQNAIGVYILPSNLTLKPALCILESHKEDFLNKFLLTISSENLKLQYKFNGQIKNPKSVNEIWTDLFSIAHVDMKLSTDRTLSSSISQFWFRLEFCKEDKDFILFPTANRYSRKLWKHSIKNNQLFKEGIRNYSEISSLPYEEDHNFDIDLVFTWVNSEDKNWQELYKKYKPDFNSDATSTSRFLSRDELKFALRSWEMNGSFIRKIFIVSNCAPPAWLDLNNPKIQWVYHEEIMPQSALPTFSSHAIETSLHHIPGISNYFIYSNDDFLLTKPLNKDNFFYSNGIAKLRLEAWGNVNGECTEGEPDYLNGARNANTLLEKEFKKFTTKLHTHSPQSMRTDILFEMEKKYPEEFNRTLHNKFRSLDDIAVTGYLYHHYALLSGRALQSSDKTELVQQNHDFKKKLNNVVTLTKERNFDKLPLSVCINDGADSHLNEEWNVQ VIKFLETLFPLPSSFEKSEQ ID NO: 10: AS ΔN69-CP-A (codon optimized) (475)LIPINFFNFEYIVKKLNNQNAIGVYILPSNLTLKPALCILESHKEDFLNKFLLTISSENLKLQYKFNGQIKNPKSVNEIWTDLFSIAHVDMKLSTDRTLSSSISQFWFRLEFCKEDKDFILFPTANRYSRKLWKHSIKNNQLFKEGIRNYSEISSLPYEEDHNFDIDLVFTWVNSEDKNWQELYKKYKPDFNSDATSTSRFLSRDELKFALRSWEMNGSFIRKIFIVSNCAPPAWLDLNNPKIQWVYHEEIMPQSALPTFSSHAIETSLHHIPGISNYFIYSNDDFLLTKPLNKDNFFYSNGIAKLRLEAWGNVNGECTEGEPDYLNGARNANTLLEKEFKKFTTKLHTHSPQSMRTDILFEMEKKYPEEFNRTLHNKFRSLDDIAVTGYLYHHYALLSGRALQSSDKTELVQQNHDFKKKLNNVVTLTKERNFDKLPLSVCINDGADSHLNEEWNVQVIKFLETLFPLPSSFEK SEQ ID NO: 11:AS ΔN97-CP-A (448) PSNLTLKPALCILESHKEDFLNKFLLTISSENLKLQYKFNGQIKNPKSVNEIWTDLFSIAHVDMKLSTDRTLSSSISQFWFRLEFCKEDKDFILFPTANRYSRKLWKHSIKNNQLFKEGIRNYSEISSLPYEEDHNFDIDLVFTWVNSEDKNWQELYKKYKPDFNSDATSTSRFLSRDELKFALRSWEMNGSFIRKIFIVSNCAPPAWLDLNNPKIQWVYHEEIMPQSALPTFSSHAIETSLHHIPGISNYFIYSNDDFLLTKPLNKDNFFYSNGIAKLRLEAWGNVNGECTEGEPDYLNGARNANTLLEKEFKKFTTKLHTHSPQSMRTDILFEMEKKYPEEFNRTLHNKFRSLDDIAVTGYLYHHYALLSGRALQSSDKTELVQQNHDFKKKLNNVVTLTKERNFDKLPLSVCINDGADSHLNEEWNVQV IKFLETLFPLPSSFEKSEQ ID NO: 12: AS ΔN167-CP-A (378)TLSSSISQFWFRLEFCKEDKDFILFPTANRYSRKLWKHSIKNNQLFKEGIRNYSEISSLPYEEDHNFDIDLVFTWVNSEDKNWQELYKKYKPDFNSDATSTSRFLSRDELKFALRSWEMNGSFIRKIFIVSNCAPPAWLDLNNPKIQWVYHEEIMPQSALPTFSSHAIETSLHHIPGISNYFIYSNDDFLLTKPLNKDNFFYSNGIAKLRLEAWGNVNGECTEGEPDYLNGARNANTLLEKEFKKFTTKLHTHSPQSMRTDILFEMEKKYPEEFNRTLHNKFRSLDDIAVTGYLYHHYALLSGRALQSSDKTELVQQNHDFKKKLNNVVTLTKERNFDKLPLSVCINDGADSHLNEEWNVQVIKFLETLFPLPSSFEK SEQ ID NO: 13:AS ΔN235-CP-A (311) DIDLVFTWVNSEDKNWQELYKKYKPDFNSDATSTSRFLSRDELKFALRSWEMNGSFIRKIFIVSNCAPPAWLDLNNPKIQWVYHEEIMPQSALPTFSSHAIETSLHHIPGISNYFIYSNDDFLLTKPLNKDNFFYSNGIAKLRLEAWGNVNGECTEGEPDYLNGARNANTLLEKEFKKFTTKLHTHSPQSMRTDILFEMEKKYPEEFNRTLHNKFRSLDDIAVTGYLYHHYALLSGRALQSSDKTELVQQNHDFKKKLNNVVTLTKERNFDKLPLSVCINDGADSHLN EEWNVQVIKFLETLFPLPSSFEKSEQ ID NO: 14: AS ΔC45-CP-A (499)FILNNRKWRKLKRDPSAFFRDSKFNFLRYFSAKKFAKNFKNSSHIHKTNISKAQSNISSTLKQNRKQDMLIPINFFNFEYIVKKLNNQNAIGVYILPSNLTLKPALCILESHKEDFLNKFLLTISSENLKLQYKFNGQIKNPKSVNEIWTDLFSIAHVDMKLSTDRTLSSSISQFWFRLEFCKEDKDFILFPTANRYSRKLWKHSIKNNQLFKEGIRNYSEISSLPYEEDHNFDIDLVFTWVNSEDKNWQELYKKYKPDFNSDATSTSRFLSRDELKFALRSWEMNGSFIRKIFIVSNCAPPAWLDLNNPKIQWVYHEEIMPQSALPTFSSHAIETSLHHIPGISNYFIYSNDDFLLTKPLNKDNFFYSNGIAKLRLEAWGNVNGECTEGEPDYLNGARNANTLLEKEFKKFTTKLHTHSPQSMRTDILFEMEKKYPEEFNRTLHNKFRSLDDIAVTGYLYHHYALLSGRALQSSDKTEL VQQNHDFKKKLNNVVTLTKSEQ ID NO: 15: AS ΔC25-CP-A (519)FILNNRKWRKLKRDPSAFFRDSKFNFLRYFSAKKFAKNFKNSSHIHKTNISKAQSNISSTLKQNRKQDMLIPINFFNFEYIVKKLNNQNAIGVYILPSNLTLKPALCILESHKEDFLNKFLLTISSENLKLQYKFNGQIKNPKSVNEIWTDLFSIAHVDMKLSTDRTLSSSISQFWFRLEFCKEDKDFILFPTANRYSRKLWKHSIKNNQLFKEGIRNYSEISSLPYEEDHNFDIDLVFTWVNSEDKNWQELYKKYKPDFNSDATSTSRFLSRDELKFALRSWEMNGSFIRKIFIVSNCAPPAWLDLNNPKIQWVYHEEIMPQSALPTFSSHAIETSLHHIPGISNYFIYSNDDFLLTKPLNKDNFFYSNGIAKLRLEAWGNVNGECTEGEPDYLNGARNANTLLEKEFKKFTTKLHTHSPQSMRTDILFEMEKKYPEEFNRTLHNKFRSLDDIAVTGYLYHHYALLSGRALQSSDKTELVQQNHDFKKKLNNVVTLTKERNFDKLPLSVCINDGADSH SEQ ID NO: 16:DNA CP-X/CsxA full-length (1455)ATTATGAGCAAAATTAGCAAATTGGTAACCCACCCAAACCTTTTCTTTCGAGATTATTTCTTAAAAAAAGCACCGTTAAATTATGGCGAAAATATTAAACCTTTACCAGTCGAAACCTCTTCTCATAGCAAAAAAAATACAGCCCATAAAACACCCGTATCATCCGACCAACCAATTGAAGATCCATACCCAGTAACATTTCCAATTGATGTAGTTTATACTTGGGTAGATTCAGATGATGAAAAATTCAATGAAGAACGCCTAAAGTTTCAAAATTCAAGCACATCTGAGACTCTACAAGGCAAAGCAGAAAGCACCGATATTGCAAGATTCCAATCACGCGACGAATTAAAATATTCGATTCGAAGCCTGATGAAGTATGCCCCATGGGTAAATCATATTTACATTGTAACAAATGGTCAAATACCAAAATGGTTAGATACCAACAATACAAAGGTAACGATTATCCCTCACTCAACTATTATCGACAGTCAATTTCTCCCTACTTTTAATTCTCACGTCATTGAATCCTCTCTATATAAAATCCCAGGATTATCAGAGCATTACATTTATTTCAATGATGATGTCATGCTAGCTAGAGATTTAAGCCCATCTTATTTCTTTACAAGCAGCGGATTAGCAAAACTGTTTATTACCAACTCTCGTCTACCAAATGGCTATAAGAATGTGAAAGACACACCAACCCAATGGGCCTCAAAAAATTCCCGTGAGCTTTTACATGCAGAAACAGGATTTTGGGCTGAAGCCATGTTTGCACATACTTTTCATCCACAACGTAAAAGTGTACATGAATCTATTGAACACCTATGGCATGAACAATTAAATGTTTGTCGTCAAAACCGTTTCCGTGATATTTCAGATATTAACATGGCGACATTCCTGCACCACCATTTTGCCATTTTGACAGGCCAAGCTCTTGCTACACGCACTAAATGTATTTACTTTAACATTCGCTCTCCTCAAGCAGCTCAGCATTACAAAACATTATTAGCTCGAAAAGGAAGCGAATACAGCCCACATTCTATCTGCTTAAATGATCATACATCGAGCAATAAAAATATTTTATCTAATTACGAAGCCAAATTACAAAGCTTTTTAGAAACATACTATCCAGATGTATCAGAAGCAGAAATTCTCCTTCCTACTAAATCTGAAGTAGCTGAATTAGTTAAACATAAAGATTATTTAACTGTATATACTAAATTATTACCTATTATCAATAAGCAGCTGGTCAATAAATATAATAAACCTTATTCATATCTTTTCTATTATTTAGGTTTATCTGCCCGGTTTTTATTTGAAGAAACGCAACAAGAACACTACCGGGAAACTGCTGAAGAAAATTTACAAATCTTTTGTGGCCTAAACCCAAAACATACACTAGCCCTCAAATACTTAGCGGATGTCACCCTCACATCA CAGCCTAGTGGACAASEQ ID NO: 17: DNA ΔN58-CP-X (1284)CCAATTGAAGATCCATACCCAGTAACATTTCCAATTGATGTAGTTTATACTTGGGTAGATTCAGATGATGAAAAATTCAATGAAGAACGCCTAAAGTTTCAAAATTCAAGCACATCTGAGACTCTACAAGGCAAAGCAGAAAGCACCGATATTGCAAGATTCCAATCACGCGACGAATTAAAATATTCGATTCGAAGCCTGATGAAGTATGCCCCATGGGTAAATCATATTTACATTGTAACAAATGGTCAAATACCAAAATGGTTAGATACCAACAATACAAAGGTAACGATTATCCCTCACTCAACTATTATCGACAGTCAATTTCTCCCTACTTTTAATTCTCACGTCATTGAATCCTCTCTATATAAAATCCCAGGATTATCAGAGCATTACATTTATTTCAATGATGATGTCATGCTAGCTAGAGATTTAAGCCCATCTTATTTCTTTACAAGCAGCGGATTAGCAAAACTGTTTATTACCAACTCTCGTCTACCAAATGGCTATAAGAATGTGAAAGACACACCAACCCAATGGGCCTCAAAAAATTCCCGTGAGCTTTTACATGCAGAAACAGGATTTTGGGCTGAAGCCATGTTTGCACATACTTTTCATCCACAACGTAAAAGTGTACATGAATCTATTGAACACCTATGGCATGAACAATTAAATGTTTGTCGTCAAAACCGTTTCCGTGATATTTCAGATATTAACATGGCGACATTCCTGCACCACCATTTTGCCATTTTGACAGGCCAAGCTCTTGCTACACGCACTAAATGTATTTACTTTAACATTCGCTCTCCTCAAGCAGCTCAGCATTACAAAACATTATTAGCTCGAAAAGGAAGCGAATACAGCCCACATTCTATCTGCTTAAATGATCATACATCGAGCAATAAAAATATTTTATCTAATTACGAAGCCAAATTACAAAGCTTTTTAGAAACATACTATCCAGATGTATCAGAAGCAGAAATTCTCCTTCCTACTAAATCTGAAGTAGCTGAATTAGTTAAACATAAAGATTATTTAACTGTATATACTAAATTATTACCTATTATCAATAAGCAGCTGGTCAATAAATATAATAAACCTTATTCATATCTTTTCTATTATTTAGGTTTATCTGCCCGGTTTTTATTTGAAGAAACGCAACAAGAACACTACCGGGAAACTGCTGAAGAAAATTTACAAATCTTTTGTGGCCTAAACCCAAAACATACACTAGCCCTCAAATACTTAGCGGATGTCACCCTCACATCACAGCCTAGTGGACAA SEQ ID NO: 18:DNA ΔN104-CP-X (1146) GAAAGCACCGATATTGCAAGATTCCAATCACGCGACGAATTAAAATATTCGATTCGAAGCCTGATGAAGTATGCCCCATGGGTAAATCATATTTACATTGTAACAAATGGTCAAATACCAAAATGGTTAGATACCAACAATACAAAGGTAACGATTATCCCTCACTCAACTATTATCGACAGTCAATTTCTCCCTACTTTTAATTCTCACGTCATTGAATCCTCTCTATATAAAATCCCAGGATTATCAGAGCATTACATTTATTTCAATGATGATGTCATGCTAGCTAGAGATTTAAGCCCATCTTATTTCTTTACAAGCAGCGGATTAGCAAAACTGTTTATTACCAACTCTCGTCTACCAAATGGCTATAAGAATGTGAAAGACACACCAACCCAATGGGCCTCAAAAAATTCCCGTGAGCTTTTACATGCAGAAACAGGATTTTGGGCTGAAGCCATGTTTGCACATACTTTTCATCCACAACGTAAAAGTGTACATGAATCTATTGAACACCTATGGCATGAACAATTAAATGTTTGTCGTCAAAACCGTTTCCGTGATATTTCAGATATTAACATGGCGACATTCCTGCACCACCATTTTGCCATTTTGACAGGCCAAGCTCTTGCTACACGCACTAAATGTATTTACTTTAACATTCGCTCTCCTCAAGCAGCTCAGCATTACAAAACATTATTAGCTCGAAAAGGAAGCGAATACAGCCCACATTCTATCTGCTTAAATGATCATACATCGAGCAATAAAAATATTTTATCTAATTACGAAGCCAAATTACAAAGCTTTTTAGAAACATACTATCCAGATGTATCAGAAGCAGAAATTCTCCTTCCTACTAAATCTGAAGTAGCTGAATTAGTTAAACATAAAGATTATTTAACTGTATATACTAAATTATTACCTATTATCAATAAGCAGCTGGTCAATAAATATAATAAACCTTATTCATATCTTTTCTATTATTTAGGTTTATCTGCCCGGTTTTTATTTGAAGAAACGCAACAAGAACACTACCGGGAAACTGCTGAAGAAAATTTACAAATCTTTTGTGGCCTAAACCCAAAACATACACTAGCCCTCAAATACTTAGCGGATGTCACCCTCACATCACAGCCTAGTGGACAA SEQ ID NO: 19:DNA ΔC174-CP-X (933) ATTATGAGCAAAATTAGCAAATTGGTAACCCACCCAAACCTTTTCTTTCGAGATTATTTCTTAAAAAAAGCACCGTTAAATTATGGCGAAAATATTAAACCTTTACCAGTCGAAACCTCTTCTCATAGCAAAAAAAATACAGCCCATAAAACACCCGTATCATCCGACCAACCAATTGAAGATCCATACCCAGTAACATTTCCAATTGATGTAGTTTATACTTGGGTAGATTCAGATGATGAAAAATTCAATGAAGAACGCCTAAAGTTTCAAAATTCAAGCACATCTGAGACTCTACAAGGCAAAGCAGAAAGCACCGATATTGCAAGATTCCAATCACGCGACGAATTAAAATATTCGATTCGAAGCCTGATGAAGTATGCCCCATGGGTAAATCATATTTACATTGTAACAAATGGTCAAATACCAAAATGGTTAGATACCAACAATACAAAGGTAACGATTATCCCTCACTCAACTATTATCGACAGTCAATTTCTCCCTACTTTTAATTCTCACGTCATTGAATCCTCTCTATATAAAATCCCAGGATTATCAGAGCATTACATTTATTTCAATGATGATGTCATGCTAGCTAGAGATTTAAGCCCATCTTATTTCTTTACAAGCAGCGGATTAGCAAAACTGTTTATTACCAACTCTCGTCTACCAAATGGCTATAAGAATGTGAAAGACACACCAACCCAATGGGCCTCAAAAAATTCCCGTGAGCTTTTACATGCAGAAACAGGATTTTGGGCTGAAGCCATGTTTGCACATACTTTTCATCCACAACGTAAAAGTGTACATGAATCTATTGAACACCTATGGCATGAACAATTAAATGTTTGTCGTCAAAACCGTTTCCGTGATATTTCAGATATTAACATGGCGACATTCCTGCACCACCAT TTTGCCATTTTGACAGGCCAASEQ ID NO: 20: DNA ΔC99-CP-X (1158)ATTATGAGCAAAATTAGCAAATTGGTAACCCACCCAAACCTTTTCTTTCGAGATTATTTCTTAAAAAAAGCACCGTTAAATTATGGCGAAAATATTAAACCTTTACCAGTCGAAACCTCTTCTCATAGCAAAAAAAATACAGCCCATAAAACACCCGTATCATCCGACCAACCAATTGAAGATCCATACCCAGTAACATTTCCAATTGATGTAGTTTATACTTGGGTAGATTCAGATGATGAAAAATTCAATGAAGAACGCCTAAAGTTTCAAAATTCAAGCACATCTGAGACTCTACAAGGCAAAGCAGAAAGCACCGATATTGCAAGATTCCAATCACGCGACGAATTAAAATATTCGATTCGAAGCCTGATGAAGTATGCCCCATGGGTAAATCATATTTACATTGTAACAAATGGTCAAATACCAAAATGGTTAGATACCAACAATACAAAGGTAACGATTATCCCTCACTCAACTATTATCGACAGTCAATTTCTCCCTACTTTTAATTCTCACGTCATTGAATCCTCTCTATATAAAATCCCAGGATTATCAGAGCATTACATTTATTTCAATGATGATGTCATGCTAGCTAGAGATTTAAGCCCATCTTATTTCTTTACAAGCAGCGGATTAGCAAAACTGTTTATTACCAACTCTCGTCTACCAAATGGCTATAAGAATGTGAAAGACACACCAACCCAATGGGCCTCAAAAAATTCCCGTGAGCTTTTACATGCAGAAACAGGATTTTGGGCTGAAGCCATGTTTGCACATACTTTTCATCCACAACGTAAAAGTGTACATGAATCTATTGAACACCTATGGCATGAACAATTAAATGTTTGTCGTCAAAACCGTTTCCGTGATATTTCAGATATTAACATGGCGACATTCCTGCACCACCATTTTGCCATTTTGACAGGCCAAGCTCTTGCTACACGCACTAAATGTATTTACTTTAACATTCGCTCTCCTCAAGCAGCTCAGCATTACAAAACATTATTAGCTCGAAAAGGAAGCGAATACAGCCCACATTCTATCTGCTTAAATGATCATACATCGAGCAATAAAAATATTTTATCTAATTACGAAGCCAAATTACAAAGCTTTTTAGAAACATACTATCCAGATGTATCAGAAGCAGAA ATTCTC SEQ ID NO: 21:DNA ΔN58ΔC99-CP-X (987) CCAATTGAAGATCCATACCCAGTAACATTTCCAATTGATGTAGTTTATACTTGGGTAGATTCAGATGATGAAAAATTCAATGAAGAACGCCTAAAGTTTCAAAATTCAAGCACATCTGAGACTCTACAAGGCAAAGCAGAAAGCACCGATATTGCAAGATTCCAATCACGCGACGAATTAAAATATTCGATTCGAAGCCTGATGAAGTATGCCCCATGGGTAAATCATATTTACATTGTAACAAATGGTCAAATACCAAAATGGTTAGATACCAACAATACAAAGGTAACGATTATCCCTCACTCAACTATTATCGACAGTCAATTTCTCCCTACTTTTAATTCTCACGTCATTGAATCCTCTCTATATAAAATCCCAGGATTATCAGAGCATTACATTTATTTCAATGATGATGTCATGCTAGCTAGAGATTTAAGCCCATCTTATTTCTTTACAAGCAGCGGATTAGCAAAACTGTTTATTACCAACTCTCGTCTACCAAATGGCTATAAGAATGTGAAAGACACACCAACCCAATGGGCCTCAAAAAATTCCCGTGAGCTTTTACATGCAGAAACAGGATTTTGGGCTGAAGCCATGTTTGCACATACTTTTCATCCACAACGTAAAAGTGTACATGAATCTATTGAACACCTATGGCATGAACAATTAAATGTTTGTCGTCAAAACCGTTTCCGTGATATTTCAGATATTAACATGGCGACATTCCTGCACCACCATTTTGCCATTTTGACAGGCCAAGCTCTTGCTACACGCACTAAATGTATTTACTTTAACATTCGCTCTCCTCAAGCAGCTCAGCATTACAAAACATTATTAGCTCGAAAAGGAAGCGAATACAGCCCACATTCTATCTGCTTAAATGATCATACATCGAGCAATAAAAATATTTTATCTAATTACGAAGCCAAATTACAAAGCTTTTTAGAAACATACTATCCAGATGTATCAGAAGCAGAAATTCTC SEQ ID NO: 22: DNA ΔN65ΔC10-CP-X (1233)GTAACATTTCCAATTGATGTAGTTTATACTTGGGTAGATTCAGATGATGAAAAATTCAATGAAGAACGCCTAAAGTTTCAAAATTCAAGCACATCTGAGACTCTACAAGGCAAAGCAGAAAGCACCGATATTGCAAGATTCCAATCACGCGACGAATTAAAATATTCGATTCGAAGCCTGATGAAGTATGCCCCATGGGTAAATCATATTTACATTGTAACAAATGGTCAAATACCAAAATGGTTAGATACCAACAATACAAAGGTAACGATTATCCCTCACTCAACTATTATCGACAGTCAATTTCTCCCTACTTTTAATTCTCACGTCATTGAATCCTCTCTATATAAAATCCCAGGATTATCAGAGCATTACATTTATTTCAATGATGATGTCATGCTAGCTAGAGATTTAAGCCCATCTTATTTCTTTACAAGCAGCGGATTAGCAAAACTGTTTATTACCAACTCTCGTCTACCAAATGGCTATAAGAATGTGAAAGACACACCAACCCAATGGGCCTCAAAAAATTCCCGTGAGCTTTTACATGCAGAAACAGGATTTTGGGCTGAAGCCATGTTTGCACATACTTTTCATCCACAACGTAAAAGTGTACATGAATCTATTGAACACCTATGGCATGAACAATTAAATGTTTGTCGTCAAAACCGTTTCCGTGATATTTCAGATATTAACATGGCGACATTCCTGCACCACCATTTTGCCATTTTGACAGGCCAAGCTCTTGCTACACGCACTAAATGTATTTACTTTAACATTCGCTCTCCTCAAGCAGCTCAGCATTACAAAACATTATTAGCTCGAAAAGGAAGCGAATACAGCCCACATTCTATCTGCTTAAATGATCATACATCGAGCAATAAAAATATTTTATCTAATTACGAAGCCAAATTACAAAGCTTTTTAGAAACATACTATCCAGATGTATCAGAAGCAGAAATTCTCCTTCCTACTAAATCTGAAGTAGCTGAATTAGTTAAACATAAAGATTATTTAACTGTATATACTAAATTATTACCTATTATCAATAAGCAGCTGGTCAATAAATATAATAAACCTTATTCATATCTTTTCTATTATTTAGGTTTATCTGCCCGGTTTTTATTTGAAGAAACGCAACAAGAACACTACCGGGAAACTGCTGAAGAAAATTTACAAATCTTTTGTGGCCTAAACCCAAAACATACACTAGCCCTCAAATACTTAGCGGAT SEQ ID NO: 23: DNA ΔN67ΔC99-CP-X (960)TTTCCAATTGATGTAGTTTATACTTGGGTAGATTCAGATGATGAAAAATTCAATGAAGAACGCCTAAAGTTTCAAAATTCAAGCACATCTGAGACTCTACAAGGCAAAGCAGAAAGCACCGATATTGCAAGATTCCAATCACGCGACGAATTAAAATATTCGATTCGAAGCCTGATGAAGTATGCCCCATGGGTAAATCATATTTACATTGTAACAAATGGTCAAATACCAAAATGGTTAGATACCAACAATACAAAGGTAACGATTATCCCTCACTCAACTATTATCGACAGTCAATTTCTCCCTACTTTTAATTCTCACGTCATTGAATCCTCTCTATATAAAATCCCAGGATTATCAGAGCATTACATTTATTTCAATGATGATGTCATGCTAGCTAGAGATTTAAGCCCATCTTATTTCTTTACAAGCAGCGGATTAGCAAAACTGTTTATTACCAACTCTCGTCTACCAAATGGCTATAAGAATGTGAAAGACACACCAACCCAATGGGCCTCAAAAAATTCCCGTGAGCTTTTACATGCAGAAACAGGATTTTGGGCTGAAGCCATGTTTGCACATACTTTTCATCCACAACGTAAAAGTGTACATGAATCTATTGAACACCTATGGCATGAACAATTAAATGTTTGTCGTCAAAACCGTTTCCGTGATATTTCAGATATTAACATGGCGACATTCCTGCACCACCATTTTGCCATTTTGACAGGCCAAGCTCTTGCTACACGCACTAAATGTATTTACTTTAACATTCGCTCTCCTCAAGCAGCTCAGCATTACAAAACATTATTAGCTCGAAAAGGAAGCGAATACAGCCCACATTCTATCTGCTTAAATGATCATACATCGAGCAATAAAAATATTTTATCTAATTACGAAGCCAAATTACAAAGCTTTTTAGAAACATACTATCCAGATGTATCAGAAGCAGAAATTCTC SEQ ID NO: 24:AS CP-X/CsxA full-length (485)IMSKISKLVTHPNLFFRDYFLKKAPLNYGENIKPLPVETSSHSKKNTAHKTPVSSDQPIEDPYPVTFPIDVVYTWVDSDDEKFNEERLKFQNSSTSETLQGKAESTDIARFQSRDELKYSIRSLMKYAPWVNHIYIVTNGQIPKWLDTNNTKVTIIPHSTIIDSQFLPTFNSHVIESSLYKIPGLSEHYIYFNDDVMLARDLSPSYFFTSSGLAKLFITNSRLPNGYKNVKDTPTQWASKNSRELLHAETGFWAEAMFAHTFHPQRKSVHESIEHLWHEQLNVCRQNRFRDISDINMATFLHHHFAILTGQALATRTKCIYFNIRSPQAAQHYKTLLARKGSEYSPHSICLNDHTSSNKNILSNYEAKLQSFLETYYPDVSEAEILLPTKSEVAELVKHKDYLTVYTKLLPIINKQLVNKYNKPYSYLFYYLGLSARFLFEETQQEHYRETAEENLQIFCGLNPKHTLALKYLADVTLTS QPSGQ SEQ ID NO: 25:AS ΔN58-CP-X (428) PIEDPYPVTFPIDVVYTWVDSDDEKFNEERLKFQNSSTSETLQGKAESTDIARFQSRDELKYSIRSLMKYAPWVNHIYIVTNGQIPKWLDTNNTKVTIIPHSTIIDSQFLPTFNSHVIESSLYKIPGLSEHYIYFNDDVMLARDLSPSYFFTSSGLAKLFITNSRLPNGYKNVKDTPTQWASKNSRELLHAETGFWAEAMFAHTFHPQRKSVHESIEHLWHEQLNVCRQNRFRDISDINMATFLHHHFAILTGQALATRTKCIYFNIRSPQAAQHYKTLLARKGSEYSPHSICLNDHTSSNKNILSNYEAKLQSFLETYYPDVSEAEILLPTKSEVAELVKHKDYLTVYTKLLPIINKQLVNKYNKPYSYLFYYLGLSARFLFEETQQEHYRETAEENLQIFCGLNPKHTLALKYLADVTLTSQPSGQ SEQ ID NO: 26:AS ΔN104-CP-X (382) ESTDIARFQSRDELKYSIRSLMKYAPWVNHIYIVTNGQIPKWLDTNNTKVTIIPHSTIIDSQFLPTFNSHVIESSLYKIPGLSEHYIYFNDDVMLARDLSPSYFFTSSGLAKLFITNSRLPNGYKNVKDTPTQWASKNSRELLHAETGFWAEAMFAHTFHPQRKSVHESIEHLWHEQLNVCRQNRFRDISDINMATFLHHHFAILTGQALATRTKCIYFNIRSPQAAQHYKTLLARKGSEYSPHSICLNDHTSSNKNILSNYEAKLQSFLETYYPDVSEAEILLPTKSEVAELVKHKDYLTVYTKLLPIINKQLVNKYNKPYSYLFYYLGLSARFLFEETQQEHYRETAEENLQIFCGLNPKHTLALKYLADVTLTSQPSGQ SEQ ID NO: 27:AS ΔC174-CP-X (311) IMSKISKLVTHPNLFFRDYFLKKAPLNYGENIKPLPVETSSHSKKNTAHKTPVSSDQPIEDPYPVTFPIDVVYTWVDSDDEKFNEERLKFQNSSTSETLQGKAESTDIARFQSRDELKYSIRSLMKYAPWVNHIYIVTNGQIPKWLDTNNTKVTIIPHSTIIDSQFLPTFNSHVIESSLYKIPGLSEHYIYFNDDVMLARDLSPSYFFTSSGLAKLFITNSRLPNGYKNVKDTPTQWASKNSRELLHAETGFWAEAMFAHTFHPQRKSVHESIEHLWHEQLNVCRQNR FRDISDINMATFLHHHFAILTGQSEQ ID NO: 28: AS ΔC99-CP-X (386)IMSKISKLVTHPNLFFRDYFLKKAPLNYGENIKPLPVETSSHSKKNTAHKTPVSSDQPIEDPYPVTFPIDVVYTWVDSDDEKFNEERLKFQNSSTSETLQGKAESTDIARFQSRDELKYSIRSLMKYAPWVNHIYIVTNGQIPKWLDTNNTKVTIIPHSTIIDSQFLPTFNSHVIESSLYKIPGLSEHYIYFNDDVMLARDLSPSYFFTSSGLAKLFITNSRLPNGYKNVKDTPTQWASKNSRELLHAETGFWAEAMFAHTFHPQRKSVHESIEHLWHEQLNVCRQNRFRDISDINMATFLHHHFAILTGQALATRTKCIYFNIRSPQAAQHYKTLLARKGSEYSPHSICLNDHTSSNKNILSNYEAKLQSFLETYYPDVSEAE IL SEQ ID NO: 29:AS ΔN58ΔC99-CP-X (329) PIEDPYPVTFPIDVVYTWVDSDDEKFNEERLKFQNSSTSETLQGKAESTDIARFQSRDELKYSIRSLMKYAPWVNHIYIVTNGQIPKWLDTNNTKVTIIPHSTIIDSQFLPTFNSHVIESSLYKIPGLSEHYIYFNDDVMLARDLSPSYFFTSSGLAKLFITNSRLPNGYKNVKDTPTQWASKNSRELLHAETGFWAEAMFAHTFHPQRKSVHESIEHLWHEQLNVCRQNRFRDISDINMATFLHHHFAILTGQALATRTKCIYFNIRSPQAAQHYKTLLARKGSEYSPHSICLNDHTSSNKNILSNYEAKLQSFLETYYPDVSEAEIL SEQ ID NO: 30:AS ΔN65ΔC10-CP-X (411) VTFPIDVVYTWVDSDDEKFNEERLKFQNSSTSETLQGKAESTDIARFQSRDELKYSIRSLMKYAPWVNHIYIVTNGQIPKWLDTNNTKVTIIPHSTIIDSQFLPTFNSHVIESSLYKIPGLSEHYIYFNDDVMLARDLSPSYFFTSSGLAKLFITNSRLPNGYKNVKDTPTQWASKNSRELLHAETGFWAEAMFAHTFHPQRKSVHESIEHLWHEQLNVCRQNRFRDISDINMATFLHHHFAILTGQALATRTKCIYFNIRSPQAAQHYKTLLARKGSEYSPHSICLNDHTSSNKNILSNYEAKLQSFLETYYPDVSEAEILLPTKSEVAELVKHKDYLTVYTKLLPIINKQLVNKYNKPYSYLFYYLGLSARFLFEETQQEHYRETAEENLQIFCGLNPKHTLALKYLAD SEQ ID NO: 31: AS ΔN67ΔC99-CP-X (320)FPIDVVYTWVDSDDEKFNEERLKFQNSSTSETLQGKAESTDIARFQSRDELKYSIRSLMKYAPWVNHIYIVTNGQIPKWLDTNNTKVTIIPHSTIIDSQFLPTFNSHVIESSLYKIPGLSEHYIYFNDDVMLARDLSPSYFFTSSGLAKLFITNSRLPNGYKNVKDTPTQWASKNSRELLHAETGFWAEAMFAHTFHPQRKSVHESIEHLWHEQLNVCRQNRFRDISDINMATFLHHHFAILTGQALATRTKCIYFNIRSPQAAQHYKTLLARKGSEYSPHSICLNDHTSSNKNILSNYEAKLQSFLETYYPDVSEAEIL SEQ ID NO: 32:UDP-GIcNAc-Epimerase (NmA) cloned from Neisseriameningitidis serogroup A, coding sequence >UDP-GIcNAc-Epimerase-NmA(AF019760 REGION: 479..1597)Atgaaagtcttaaccgtctttggcactcgccctgaagctattaaaatggcgcctgtaattctagagttacaaaaacataacacaattacttcaaaagtttgcattactgcacagcatcgtgaaatgctagatcaggttttgagcctattcgaaatcaaagctgattatgatttaaatatcatgaaacccaaccagagcctacaagaaatcacaacaaatatcatctcaagccttaccgatgttcttgaagatttcaaacctgactgcgtccttgctcacggagacaccacaacaacttttgcagctagccttgctgcattctatcaaaaaatacctgttggccacattgaagcaggcctgagaacttataatttatactctccttggccagaggaagcaaataggcgtttaacaagcgttctaagccagtggcattttgcacctactgaagattctaaaaataacttactatctgaatcaataccttctgacaaagttattgttactggaaatactgtcatagatgcactaatggtatctctagaaaaactaaaaataactacaattaaaaaacaaatggaacaagcttttccatttattcaggacaactctaaagtaattttaattaccgctcatagaagagaaaatcatggggaaggtattaaaaatattggactttctatcttagaattagctaaaaaatacccaacattctcttttgtgattccgctccatttaaatcctaacgttagaaaaccaattcaagatttattatcctctgtgcacaatgttcatcttattgagccacaagaatacttaccattcgtatatttaatgtctaaaagccatataatattaagtgattcaggcggcatacaagaagaagctccatccctaggaaaaccagttcttgtattaagagatactacagaacgtcctgaagctgtagctgcaggaactgtaaaattagtaggttctgaaactcaaaatattattgagagctttacacaactaattgaataccctgaatattatgaaaaaatggctaatattgaaaacccttacgggataggtaatgcctcaaaaatcattgtagaaacttta ttaaagaatagataaSEQ ID NO: 33: UDP-GIcNAc-Epimerase (NmA) cloned from Neisseria meningitidis serogroup A aminoacid sequence >UDP-GIcNAc-Epimerase-NmA (AAC38285).promkvltvfgtrpeaikmapvilelqkhntitskvcitaqhremldqvlslfeikadydlnimkpnqslgeittniissltdvledfkpdcvlahgdttttfaaslaafyqkipvghieaglrtynlyspwpeeanrrltsvlsqwhfaptedsknnllsesipsdkvivtgntvidalmvsleklkittikkqmegafpfiqdnskvilitahrrenhgegikniglsilelakkyptfsfyiplhlnpnvrkpiqdllssvhnvhliepqeylpfvylmskshiilsdsggiqeeapslgkpvlvlrdtterpeavaagtvklygsetqniiesftqlieypeyyekmanienpygignaskiivetllknr

The sequences provided herein (e.g. the sequences corresponding to CP-Aor fragments thereof) may be linked to (a) tag(s). For example, thesequences may have a StrepII-thrombin-tag at the N-terminus and/or ahis-tag at the C-terminus. The constructs may be prepared by usingappropriate restriction sites. For example, CP-A-constructs may becloned by using BamHI/BglII and XhoI. It is mentioned that, if theN-terminus is extended (e.g. by tags), then ATG/Met1 can be omitted.Accordingly, a construct comprising the full length CP-A may comprisenot 545 but 544 CP-A-amino acids. SEQ ID NOs: 34 and 37 show an examplefor a StrepII-thrombin-BamHI/BglII-CP-A-XhoI-his construct. In contextof the invention, all herein disclosed sequences may be used to produceconstructs as demonstrated for CP-A in SEQ ID NOs: 34 and 37.

SEQ ID NO: 34: DNA StrepII-Thrombin-BamHI/BgIII-CsaB-XhoI-His6atggctagctggagccacccgcagttcgaaaaaggcgccctggttccgcgtgGATCTtttatacttaataacagaaaatggcgtaaacttaaaagagaccctagcgctttctttcgagatagtaaatttaactttttaagatatttttctgctaaaaaatttgcaaagaattttaaaaattcatcacatatccataaaactaatataagtaaagctcaatcaaatatttcttcaaccttaaaacaaaatcggaaacaagatatgttaattcctattaatttttttaattttgaatatatagttaaaaaacttaacaatcaaaacgcaataggtgtatatattcttccttctaatcttactcttaagcctgcattatgtattctagaatcacataaagaagactttttaaataaatttcttcttactatttcctctgaaaatttaaagcttcaatacaaatttaatggacaaataaaaaatcctaagtccgtaaatgaaatttggacagatttatttagcattgctcatgttgacatgaaactcagcacagatagaactttaagttcatctatatctcaattttggttcagattagagttctgtaaagaagataaggattttatcttatttcctacagctaacagatattctagaaaactttggaagcactctattaaaaataatcaattatttaaagaaggcatacgaaactattcagaaatatcttcattaccctatgaagaagatcataattttgatattgatttagtatttacttgggtcaactcagaagataagaattggcaagagttatataaaaaatataagcccgactttaatagcgatgcaaccagtacatcaagattccttagtagagatgaattaaaattcgcattacgctcttgggaaatgaatggatccttcattcgaaaaatttttattgtctctaattgtgctcccccagcatggctagatttaaataaccctaaaattcaatgggtatatcacgaagaaattatgccacaaagtgcccttcctacttttagctcacatgctattgaaaccagcttgcaccatataccaggaattagtaactattttatttacagcaatgacgacttcctattaactaaaccattgaataaagacaatttcttctattcgaatggtattgcaaagttaagattagaagcatggggaaatgttaatggtgaatgtactgaaggagaacctgactacttaaatggtgctcgcaatgcgaacactctcttagaaaaggaatttaaaaaatttactactaaactacatactcactcccctcaatccatgagaactgatattttatttgagatggaaaaaaaatatccagaagagtttaatagaacactacataataaattccgatctttagatgatattgcagtaacgggctatctctatcatcattatgccctactctctggacgagcactacaaagttctgacaagacggaacttgtacagcaaaatcatgatttcaaaaagaaactaaataatgtagtgaccttaactaaagaaaggaattttgacaaacttcctttgagcgtatgtatcaacgatggtgctgatagtcacttgaatgaagaatggaatgttcaagttattaagttcttagaaactcttttcccattaccatcatcatttgagaaacTCGAGcaccaccaccaccaccac SEQ ID NO: 35:DNA StrepII-Thrombin-BamHI/BgIIIatggctagctggagccacccgcagttcgaaaaaggcgccctggttccg cgtgGATCTSEQ ID NO: 36: DNA XhoI-His6 cTCGAGcaccaccaccaccaccac SEQ ID NO: 37:AS StrepII-Thrombin-BamHI/BgIII-CsaB-XhoI-His6MASWSHPQFEKGALVPRGSFILNNRKWRKLKRDPSAFFRDSKFNFLRYFSAKKFAKNFKNSSHIHKTNISKAQSNISSTLKQNRKQD M LIPINFFNFEYIVKKLNNQNAIGVYILPSNLTLKPALCILESHKEDFLNKFLLTISSENLKLQYKFNGQIKNPKSVNEIWTDLFSIAHVDMKLSTDRTLSSSISQFWFRLEFCKEDKDFILFPTANRYSRKLWKHSIKNNQLFKEGIRNYSEISSLPYEEDHNFDIDLVFTWVNSEDKNWQELYKKYKPDFNSDATSTSRFLSRDELKFALRSWEMNGSFIRKIFIVSNCAPPAWLDLNNPKIQWVYHEEIMPQSALPTFSSHAIETSLHHIPGISNYFIYSNDDFLLTKPLNKDNFFYSNGIAKLRLEAWGNVNGECTEGEPDYLNGARNANTLLEKEFKKFTTKLHTHSPQSMRTDILFEMEKKYPEEFNRTLHNKFRSLDDIAVTGYLYHHYALLSGRALQSSDKTELVQQNHDFKKKLNNVVTLTKERNFDKLPLSVCINDGADSHLNEEWNVQVIKFLETLFPLPSSFEKLEHHHHHH SEQ ID NO: 38:AS StrepII-Thrombin-BamHI MASWSHPQFEKGALVPRGS SEQ ID NO: 39:AS XhoI-His6 LEHHHHHH

The sequences provided herein (e.g. the sequences corresponding toΔ69-CP-A) may be cloned into the vector by using NdeI/XhoI. SEQ ID NOs:40 and 43 show an example for a NdeI-ΔN69-CP-A-XhoI-his construct. Incontext of the invention, all herein disclosed sequences may be used toproduce constructs as demonstrated for the codon optimized ΔN69-CP-A inSEQ ID NOs: 40 and 43.

SEQ ID NO: 40: DNA NdeI-dN69CsaB(co)-XhoI-His6catatgCTGATCCCGATCAATTTCTTTAATTTTGAGTACATCGTGAAGAAACTGAATAACCAAAACGCAATCGGCGTGTACATTCTGCCGTCTAATCTGACCCTGAAACCAGCATTGTGCATCTTGGAGTCGCACAAAGAGGACTTCCTGAACAAATTTTTGTTGACCATTAGCAGCGAGAACCTGAAACTGCAGTATAAGTTCAATGGTCAGATCAAAAATCCGAAAAGCGTGAACGAAATCTGGACCGACCTGTTTAGCATTGCTCACGTCGACATGAAGCTGAGCACCGACCGTACGCTGTCCTCGTCCATCAGCCAATTTTGGTTTCGCCTGGAGTTCTGTAAAGAGGACAAGGACTTCATCCTGTTTCCGACGGCAAATCGTTACAGCCGCAAGCTGTGGAAGCACAGCATCAAAAATAATCAGCTGTTTAAGGAAGGTATCCGTAACTACAGCGAGATTAGCTCGCTGCCGTACGAGGAAGACCATAACTTCGACATCGATCTGGTCTTTACCTGGGTCAATTCGGAAGACAAAAACTGGCAGGAACTGTACAAGAAATATAAGCCGGATTTTAATAGCGATGCCACCTCGACGAGCCGTTTTCTGAGCCGTGACGAGCTGAAGTTTGCGCTGCGCTCGTGGGAAATGAACGGTAGCTTCATCCGTAAAATCTTTATCGTCAGCAACTGCGCGCCGCCGGCCTGGCTGGATCTGAACAATCCGAAGATCCAATGGGTGTATCACGAGGAGATCATGCCACAGAGCGCCCTGCCAACCTTCAGCAGCCATGCTATTGAGACTAGCTTGCATCACATTCCGGGCATCTCCAACTACTTCATCTACTCTAATGACGATTTTCTGTTGACCAAACCGCTGAACAAAGACAACTTCTTTTACTCCAACGGTATTGCTAAACTGCGTCTGGAAGCCTGGGGTAACGTTAACGGTGAATGTACCGAAGGCGAGCCGGATTACCTGAACGGCGCGCGTAACGCAAATACGCTGCTGGAGAAAGAGTTTAAAAAGTTTACCACCAAGCTGCACACCCACAGCCCGCAGAGCATGCGTACCGACATCCTGTTCGAGATGGAGAAAAAATACCCAGAAGAGTTCAATCGCACGCTGCACAACAAGTTCCGCAGCCTGGATGACATCGCGGTTACCGGCTACCTGTACCATCACTACGCATTGCTGTCTGGCCGCGCTCTGCAATCCAGCGATAAGACCGAACTGGTCCAGCAGAATCACGACTTTAAAAAGAAGCTGAATAATGTTGTCACCCTGACCAAAGAGCGTAACTTTGATAAGCTGCCGCTGAGCGTTTGTATTAATGACGGTGCAGACAGCCACCTGAATGAGGAGTGGAATGTGCAAGTTATCAAATTCTTGGAGACCTTGTTCCCGTTGCCGAGCTCCTTCGAGAAAcTCGAGcac caccaccaccaccacSEQ ID NO: 41: DNA NdeI catatg SEQ ID NO: 42: DNA XhoI-His6cTCGAGcaccaccaccaccaccac SEQ ID NO: 43: AS NdeI-dN69CsaB(co)-XhoI-His6MLIPINFFNFEYIVKKLNNQNAIGVYILPSNLTLKPALCILESHKEDFLNKFLLTISSENLKLQYKFNGQIKNPKSVNEIWTDLFSIAHVDMKLSTDRTLSSSISQFWFRLEFCKEDKDFILFPTANRYSRKLWKHSIKNNQLFKEGIRNYSEISSLPYEEDHNFDIDLVFTWVNSEDKNWQELYKKYKPDFNSDATSTSRFLSRDELKFALRSWEMNGSFIRKIFIVSNCAPPAWLDLNNPKIQWVYHEEIMPQSALPTFSSHAIETSLHHIPGISNYFIYSNDDFLLTKPLNKDNFFYSNGIAKLRLEAWGNVNGECTEGEPDYLNGARNANTLLEKEFKKFTTKLHTHSPQSMRTDILFEMEKKYPEEFNRTLHNKFRSLDDIAVTGYLYHHYALLSGRALQSSDKTELVQQNHDFKKKLNNVVTLTKERNFDKLPLSVCINDGADSHLNEEWNVQVIKFLETLFPLPSSFEKLEHH HHHH SEQ ID NO: 44:AS NdeI M SEQ ID NO: 45: AS XhoI-His6 LEHHHHHH SEQ ID NO: 46:DNA ΔN69-CP-A(wt)(1425) ttaattcctattaatttttttaattttgaatatatagttaaaaaacttaacaatcaaaacgcaataggtgtatatattcttccttctaatcttactcttaagcctgcattatgtattctagaatcacataaagaagactttttaaataaatttcttcttactatttcctctgaaaatttaaagcttcaatacaaatttaatggacaaataaaaaatcctaagtccgtaaatgaaatttggacagatttatttagcattgctcatgttgacatgaaactcagcacagatagaactttaagttcatctatatctcaattttggttcagattagagttctgtaaagaagataaggattttatcttatttcctacagctaacagatattctagaaaactttggaagcactctattaaaaataatcaattatttaaagaaggcatacgaaactattcagaaatatcttcattaccctatgaagaagatcataattttgatattgatttagtatttacttgggtcaactcagaagataagaattggcaagagttatataaaaaatataagcccgactttaatagcgatgcaaccagtacatcaagattccttagtagagatgaattaaaattcgcattacgctcttgggaaatgaatggatccttcattcgaaaaatttttattgtctctaattgtgctcccccagcatggctagatttaaataaccctaaaattcaatgggtatatcacgaagaaattatgccacaaagtgcccttcctacttttagctcacatgctattgaaaccagcttgcaccatataccaggaattagtaactattttatttacagcaatgacgacttcctattaactaaaccattgaataaagacaatttcttctattcgaatggtattgcaaagttaagattagaagcatggggaaatgttaatggtgaatgtactgaaggagaacctgactacttaaatggtgctcgcaatgcgaacactctcttagaaaaggaatttaaaaaatttactactaaactacatactcactcccctcaatccatgagaactgatattttatttgagatggaaaaaaaatatccagaagagtttaatagaacactacataataaattccgatctttagatgatattgcagtaacgggctatctctatcatcattatgccctactctctggacgagcactacaaagttctgacaagacggaacttgtacagcaaaatcatgatttcaaaaagaaactaaataatgtagtgaccttaactaaagaaaggaattttgacaaacttcctttgagcgtatgtatcaacgatggtgctgatagtcacttgaatgaagaatggaatgttcaagttattaagttcttagaaactcttttcccattaccatcatcatttgagaaa

The sequences provided herein (e.g. the sequences corresponding to CP-Xor fragments thereof) may be linked to (a) tag(s). For example, thesequences may have an N-terminal MBP-tag followed by the proteaseresistant linker S3N10, and/or a C-terminal his-tag. The sequencesprovided herein (e.g. the sequences corresponding to CP-X or fragmentsthereof) may be cloned into the vector by using the restriction sitesBamHI and/or XhoI. In particular in the constructs of CP-X (or fragmentsthereof), if the N-terminus is not truncated, then the ATG1/Met1 may notbe in the construct. SEQ ID NOs: 47 and 49 show an example for aMBP-S3N10-BamHI-CP-X-XhoI-his construct. In context of the invention,all herein disclosed sequences may be used to produce constructs asdemonstrated for CP-X in SEQ ID NOs: 47 and 49.

SEQ ID NO: 47: DNA MBP-S3N10-BamHI-CsxA-Xho1-His6atgaaaactgaagaaggtaaactggtaatctggattaacggcgataaaggctataacggtctcgctgaagtcggtaagaaattcgagaaagataccggaattaaagtcaccgttgagcatccggataaactggaagagaaattcccacaggttgcggcaactggcgatggccctgacattatcttctgggcacacgaccgctttggtggctacgctcaatctggcctgttggctgaaatcaccccggacaaagcgttccaggacaagctgtatccgtttacctgggatgccgtacgttacaacggcaagctgattgcttacccgatcgctgttgaagcgttatcgctgatttataacaaagatctgctgccgaacccgccaaaaacctgggaagagatcccggcgctggataaagaactgaaagcgaaaggtaagagcgcgctgatgttcaacctgcaagaaccgtacttcacctggccgctgattgctgctgacgggggttatgcgttcaagtatgaaaacggcaagtacgacattaaagacgtgggcgtggataacgctggcgcgaaagcgggtctgaccttcctggttgacctgattaaaaacaaacacatgaatgcagacaccgattactccatcgcagaagctgcctttaataaaggcgaaacagcgatgaccatcaacggcccgtgggcatggtccaacatcgacaccagcaaagtgaattatggtgtaacggtactgccgaccttcaagggtcaaccatccaaaccgttcgttggcgtgctgagcgcaggtattaacgccgccagtccgaacaaagagctggcgaaagagttcctcgaaaactatctgctgactgatgaaggtctggaagcggttaataaagacaaaccgctgggtgccgtagcgctgaagtcttacgaggaagagttggcgaaagatccacgtattgccgccaccatggaaaacgcccagaaaggtgaaatcatgccgaacatcccgcagatgtccgctttctggtatgccgtgcgtactgcggtgatcaacgccgccagcggtcgtcagactgtcgatgaagccctgaaagacgcgcagactaatTCGAGCTCCAATAACAATAACAACAACAATAAcAATAACGGATCCATTATGAGCAAAATTAGCAAATTGGTAACCCACCCAAACCTTTTCTTTCGAGATTATTTCTTAAAAAAAGCACCGTTAAATTATGGCGAAAATATTAAACCTTTACCAGTCGAAACCTCTTCTCATAGCAAAAAAAATACAGCCCATAAAACACCCGTATCATCCGACCAACCAATTGAAGATCCATACCCAGTAACATTTCCAATTGATGTAGTTTATACTTGGGTAGATTCAGATGATGAAAAATTCAATGAAGAACGCCTAAAGTTTCAAAATTCAAGCACATCTGAGACTCTACAAGGCAAAGCAGAAAGCACCGATATTGCAAGATTCCAATCACGCGACGAATTAAAATATTCGATTCGAAGCCTGATGAAGTATGCCCCATGGGTAAATCATATTTACATTGTAACAAATGGTCAAATACCAAAATGGTTAGATACCAACAATACAAAGGTAACGATTATCCCTCACTCAACTATTATCGACAGTCAATTTCTCCCTACTTTTAATTCTCACGTCATTGAATCCTCTCTATATAAAATCCCAGGATTATCAGAGCATTACATTTATTTCAATGATGATGTCATGCTAGCTAGAGATTTAAGCCCATCTTATTTCTTTACAAGCAGCGGATTAGCAAAACTGTTTATTACCAACTCTCGTCTACCAAATGGCTATAAGAATGTGAAAGACACACCAACCCAATGGGCCTCAAAAAATTCCCGTGAGCTTTTACATGCAGAAACAGGATTTTGGGCTGAAGCCATGTTTGCACATACTTTTCATCCACAACGTAAAAGTGTACATGAATCTATTGAACACCTATGGCATGAACAATTAAATGTTTGTCGTCAAAACCGTTTCCGTGATATTTCAGATATTAACATGGCGACATTCCTGCACCACCATTTTGCCATTTTGACAGGCCAAGCTCTTGCTACACGCACTAAATGTATTTACTTTAACATTCGCTCTCCTCAAGCAGCTCAGCATTACAAAACATTATTAGCTCGAAAAGGAAGCGAATACAGCCCACATTCTATCTGCTTAAATGATCATACATCGAGCAATAAAAATATTTTATCTAATTACGAAGCCAAATTACAAAGCTTTTTAGAAACATACTATCCAGATGTATCAGAAGCAGAAATTCTCCTTCCTACTAAATCTGAAGTAGCTGAATTAGTTAAACATAAAGATTATTTAACTGTATATACTAAATTATTACCTATTATCAATAAGCAGCTGGTCAATAAATATAATAAACCTTATTCATATCTTTTCTATTATTTAGGTTTATCTGCCCGGTTTTTATTTGAAGAAACGCAACAAGAACACTACCGGGAAACTGCTGAAGAAAATTTACAAATCTTTTGTGGCCTAAACCCAAAACATACACTAGCCCTCAAATACTTAGCGGATGTCACCCTCACATCACAGCCTAGTGGACAACtcgagcaccaccaccaccaccac SEQ ID NO: 48:DNA MBP-S3N10-BamHI atgaaaactgaagaaggtaaactggtaatctggattaacggcgataaaggctataacggtctcgctgaagtcggtaagaaattcgagaaagataccggaattaaagtcaccgttgagcatccggataaactggaagagaaattcccacaggttgcggcaactggcgatggccctgacattatcttctgggcacacgaccgctttggtggctacgctcaatctggcctgttggctgaaatcaccccggacaaagcgttccaggacaagctgtatccgtttacctgggatgccgtacgttacaacggcaagctgattgcttacccgatcgctgttgaagcgttatcgctgatttataacaaagatctgctgccgaacccgccaaaaacctgggaagagatcccggcgctggataaagaactgaaagcgaaaggtaagagcgcgctgatgttcaacctgcaagaaccgtacttcacctggccgctgattgctgctgacgggggttatgcgttcaagtatgaaaacggcaagtacgacattaaagacgtgggcgtggataacgctggcgcgaaagcgggtctgaccttcctggttgacctgattaaaaacaaacacatgaatgcagacaccgattactccatcgcagaagctgcctttaataaaggcgaaacagcgatgaccatcaacggcccgtgggcatggtccaacatcgacaccagcaaagtgaattatggtgtaacggtactgccgaccttcaagggtcaaccatccaaaccgttcgttggcgtgctgagcgcaggtattaacgccgccagtccgaacaaagagctggcgaaagagttcctcgaaaactatctgctgactgatgaaggtctggaagcggttaataaagacaaaccgctgggtgccgtagcgctgaagtcttacgaggaagagttggcgaaagatccacgtattgccgccaccatggaaaacgcccagaaaggtgaaatcatgccgaacatcccgcagatgtccgctttctggtatgccgtgcgtactgcggtgatcaacgccgccagcggtcgtcagactgtcgatgaagccctgaaagacgcgcagactaatTCGAGCTCCAATAACAATAACAACAACAATAAcAATAACGGATCC SEQ ID NO: 49:AS MBP-S3N10-BamHI-CsxA-Xho1-His6MKTEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLIAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGETAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAFWYAVRTAVINAASGRQTVDEALKDAQTNSSSNNNNNNNNNNGSIMSKISKLVTHPNLFFRDYFLKKAPLNYGENIKPLPVETSSHSKKNTAHKTPVSSDQPIEDPYPVTFPIDVVYTWVDSDDEKFNEERLKFQNSSTSETLQGKAESTDIARFQSRDELKYSIRSLMKYAPWVNHIYIVTNGQIPKWLDTNNTKVTIIPHSTIIDSQFLPTFNSHVIESSLYKIPGLSEHYIYFNDDVMLARDLSPSYFFTSSGLAKLFITNSRLPNGYKNVKDTPTQWASKNSRELLHAETGFWAEAMFAHTFHPQRKSVHESIEHLWHEQLNVCRQNRFRDISDINMATFLHHHFAILTGQALATRTKCIYFNIRSPQAAQHYKTLLARKGSEYSPHSICLNDHTSSNKNILSNYEAKLQSFLETYYPDVSEAEILLPTKSEVAELVKHKDYLTVYTKLLPIINKQLVNKYNKPYSYLFYYLGLSARFLFEETQQEHYRETAEENLQIFCGLNPKHTLALKYLADVTLTSQ PSGQLEHHHHHHSEQ ID NO: 50: AS MBP-S3N10-BamHIMKTEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLIAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGETAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAFWYAVRTAVINAASGRQTVDEALKDAQTNSSSNNNNNNNNNNGS

The sequences provided herein (e.g. the sequences corresponding toΔN65ΔC10-CP-X) may have a free N-terminus. These constructs may be used,inter alia for crystallization approaches. For example, the sequencesmay be cloned into a vector by using NdeI and/or XhoI. SEQ ID NOs: 51and 52 show an example for an NdeI-ΔN65ΔC10-CP-X-XhoI-his construct. Incontext of the invention, all herein disclosed sequences may be used toproduce constructs as demonstrated for ΔN65ΔC10-CP-X in SEQ ID NOs: 51and 52.

SEQ ID NO: 51: DNA NdeI-dN65dC10-CsxA-XhoI-His6caTATGGTAACATTTCCAATTGATGTAGTTTATACTTGGGTAGATTCAGATGATGAAAAATTCAATGAAGAACGCCTAAAGTTTCAAAATTCAAGCACATCTGAGACTCTACAAGGCAAAGCAGAAAGCACCGATATTGCAAGATTCCAATCACGCGACGAATTAAAATATTCGATTCGAAGCCTGATGAAGTATGCCCCATGGGTAAATCATATTTACATTGTAACAAATGGTCAAATACCAAAATGGTTAGATACCAACAATACAAAGGTAACGATTATCCCTCACTCAACTATTATCGACAGTCAATTTCTCCCTACTTTTAATTCTCACGTCATTGAATCCTCTCTATATAAAATCCCAGGATTATCAGAGCATTACATTTATTTCAATGATGATGTCATGCTAGCTAGAGATTTAAGCCCATCTTATTTCTTTACAAGCAGCGGATTAGCAAAACTGTTTATTACCAACTCTCGTCTACCAAATGGCTATAAGAATGTGAAAGACACACCAACCCAATGGGCCTCAAAAAATTCCCGTGAGCTTTTACATGCAGAAACAGGATTTTGGGCTGAAGCCATGTTTGCACATACTTTTCATCCACAACGTAAAAGTGTACATGAATCTATTGAACACCTATGGCATGAACAATTAAATGTTTGTCGTCAAAACCGTTTCCGTGATATTTCAGATATTAACATGGCGACATTCCTGCACCACCATTTTGCCATTTTGACAGGCCAAGCTCTTGCTACACGCACTAAATGTATTTACTTTAACATTCGCTCTCCTCAAGCAGCTCAGCATTACAAAACATTATTAGCTCGAAAAGGAAGCGAATACAGCCCACATTCTATCTGCTTAAATGATCATACATCGAGCAATAAAAATATTTTATCTAATTACGAAGCCAAATTACAAAGCTTTTTAGAAACATACTATCCAGATGTATCAGAAGCAGAAATTCTCCTTCCTACTAAATCTGAAGTAGCTGAATTAGTTAAACATAAAGATTATTTAACTGTATATACTAAATTATTACCTATTATCAATAAGCAGCTGGTCAATAAATATAATAAACCTTATTCATATCTTTTCTATTATTTAGGTTTATCTGCCCGGTTTTTATTTGAAGAAACGCAACAAGAACACTACCGGGAAACTGCTGAAGAAAATTTACAAATCTTTTGTGGCCTAAACCCAAAACATACACTAGCCCTCAAATACTTAGCGGATctcgagcac caccaccaccaccacSEQ ID NO: 52: AS NdeI-dN65dC10-CsxA-XhoI-His6MVTFPIDVVYTWVDSDDEKFNEERLKFQNSSTSETLQGKAESTDIARFQSRDELKYSIRSLMKYAPWVNHIYIVTNGQIPKWLDTNNTKVTIIPHSTIIDSQFLPTFNSHVIESSLYKIPGLSEHYIYFNDDVMLARDLSPSYFFTSSGLAKLFITNSRLPNGYKNVKDTPTQWASKNSRELLHAETGFWAEAMFAHTFHPQRKSVHESIEHLWHEQLNVCRQNRFRDISDINMATFLHHHFAILTGQALATRTKCIYFNIRSPQAAQHYKTLLARKGSEYSPHSICLNDHTSSNKNILSNYEAKLQSFLETYYPDVSEAEILLPTKSEVAELVKHKDYLTVYTKLLPIINKQLVNKYNKPYSYLFYYLGLSARFLFEETQQEHYRETAEENLQIFCGLNPKHTLALKYLADLEHHHHHH SEQ ID NO: 53: DNA CsaCAtgttatctaatttaaaaacaggaaataatatcttaggattacctgaatttgagttgaatggctgccgattcttatataaaaaaggtatagaaaaaacaattattactttttcagcatttcctcctaaagatattgctcaaaaatataattatataaaagattttttaagttctaattatacttttttagcattcttagataccaaatatccagaagatgatgctagaggcacttattacattactaatgagttagataatggatatttacaaaccatacattgtattattcaattattatcgaatacaaatcaagaagatacctaccttttgggttcaagtaaaggtggcgttggcgcacttctactcggtcttacatataattatcctaatataattattaatgctcctcaagccaaattagcagattatatcaaaacacgctcgaaaaccattctttcatatatgcttggaacctctaaaagatttcaagatattaattacgattatatcaatgacttcttactatctaaaattaagacttgcgactcctcacttaaatggaatattcatataacttgcggaaaagatgattcatatcatttaaatgaattagaaattctaaaaaatgaatttaatataaaagctattacgattaaaaccaaactaatttctggcgggcatgataatgaagcaattgcccactatagagaatacttt aaaaccataatccaaaatataSEQ ID NO: 54: AS CsaC MLSNLKTGNNILGLPEFELNGCRFLYKKGIEKTIITFSAFPPKDIAQKYNYIKDFLSSNYTFLAFLDTKYPEDDARGTYYITNELDNGYLQTIHCIIQLLSNTNQEDTYLLGSSKGGVGALLLGLTYNYPNIIINAPQAKLADYIKTRSKTILSYMLGTSKRFQDINYDYINDFLLSKIKTCDSSLKWNIHITCGKDDSYHLNELEILKNEFNIKAITIKTKLISGGHDNEAIAHYREYF KTIIQNI

1. In vitro method for producing Neisseria meningitidis capsularpolysaccharides which have a defined length, said method comprising thesteps: (a) incubating at least one capsule polymerase with at least onedonor carbohydrate and at least one acceptor carbohydrate; wherein theratio of donor carbohydrate to acceptor carbohydrate is a ratio from10:1 to 400:1; and (b) isolating the resulting capsular polysaccharides,wherein the capsule polymerase is the capsule polymerase of Neisseriameningitidis serogroup A or a truncated version of the capsulepolymerase of Neisseria meningitidis serogroup X.
 2. In vitro method forproducing Neisseria meningitidis capsular polysaccharides which have adefined length, said method comprising the steps: (a) incubating atleast one capsule polymerase with at least one donor carbohydrate and atleast one acceptor carbohydrate; wherein the incubation time ranges from3 to 45 minutes; and (b) isolating the resulting capsularpolysaccharides, wherein the capsule polymerase is a truncated versionof the capsule polymerase of Neisseria meningitidis serogroup X.
 3. Invitro method for producing Neisseria meningitidis capsularpolysaccharides which have a defined length, said method comprising thesteps: (a) incubating at least one capsule polymerase with at least onedonor carbohydrate and at least one acceptor carbohydrate; wherein (i)the ratio of donor carbohydrate to acceptor carbohydrate is a ratio from10:1 to 10000:1, and (ii) the incubation time ranges from 3 to 45minutes; and (b) isolating the resulting capsular polysaccharides,wherein the capsule polymerase is a truncated version of the capsulepolymerase of Neisseria meningitidis serogroup X.
 4. A compositioncomprising: (i) at least one capsule polymerase; (ii) at least one donorcarbohydrate; and (iii) at least one acceptor carbohydrate, wherein theratio of donor carbohydrate to acceptor carbohydrate is a ratio from10:1 to 10000:1, and wherein the capsule polymerase is the capsulepolymerase of Neisseria meningitidis serogroup A or a truncated versionof the capsule polymerase of Neisseria meningitidis serogroup X.
 5. Thecomposition of claim 4, wherein the ratio of donor carbohydrate toacceptor carbohydrate is a ratio from 20:1 to 240:1.
 6. The method ofclaim 1 or the composition of claim 4 or 5, wherein the capsulepolymerase of Neisseria meningitidis serogroup A is the polypeptide ofany one of (a) to (f): (a) a polypeptide comprising an amino acidsequence encoded by a nucleic acid molecule having the nucleic acidsequence of any one of SEQ ID NO: 1 to 3; (b) a polypeptide comprisingthe amino acid sequence of SEQ ID NO: 9 or 10; (c) a polypeptide encodedby a nucleic acid molecule encoding a polypeptide comprising the aminoacid sequence of SEQ ID NO: 9 or 10 or of a functional fragment thereof,wherein the function comprises the ability to transfer ManNAc-1P or aderivative thereof from UDP-ManNAc or a derivative thereof onto anacceptor carbohydrate; (d) a polypeptide comprising an amino acidsequence encoded by a nucleic acid molecule hybridizing under stringentconditions to the complementary strand of a nucleic acid molecule asdefined in (a) or (c) and encoding a functional polypeptide; or afunctional fragment thereof, wherein the function comprises the abilityto transfer ManNAc-1P or a derivative thereof from UDP-ManNAc or aderivative thereof onto an acceptor carbohydrate; (e) a polypeptidehaving at least 80% identity to the polypeptide of any one of (a) to(d), whereby said polypeptide is functional; or a functional fragmentthereof, wherein the function comprises the ability to transferManNAc-1P or a derivative thereof from UDP-ManNAc or a derivativethereof onto an acceptor carbohydrate; and (f) a polypeptide comprisingan amino acid sequence encoded by a nucleic acid molecule beingdegenerate as a result of the genetic code to the nucleotide sequence ofa nucleic acid molecule as defined in (a), (c), and (d).
 7. Apolypeptide comprising: (a) an amino acid sequence encoded by a nucleicacid molecule which is a terminally truncated version of the nucleicacid sequence of SEQ ID NO: 16 and comprises maximal 50-95% of thenucleic acid sequence of SEQ ID NO: 16; (b) an amino acid sequence whichis a terminally truncated version of the amino acid sequence of SEQ IDNO: 24 and comprises maximal 50-95% of the amino acid sequence of SEQ IDNO: 24; (c) a polypeptide encoded by a nucleic acid molecule encoding apolypeptide having an amino acid sequence which is a terminallytruncated version of the amino acid sequence of SEQ ID NO: 24 andcomprises maximal 50-95% of the amino acid sequence of SEQ ID NO: 24;(d) a polypeptide encoded by a nucleic acid molecule hybridizing understringent conditions to the complementary strand of a nucleic acidmolecule as defined in (a) or (c) and encoding a functional polypeptide;wherein the function comprises the ability to transfer GlcNAc-1P or aderivative thereof from UDP-GlcNAc or a derivative thereof onto anacceptor carbohydrate; (e) a polypeptide having at least 80% identity tothe polypeptide of any one of (a) to (d), whereby said polypeptide isfunctional; wherein the function comprises the ability to transferGlcNAc-1P or a derivative thereof from UDP-GlcNAc or a derivativethereof onto an acceptor carbohydrate; or (f) a polypeptide comprisingan amino acid sequence encoded by a nucleic acid molecule beingdegenerate as a result of the genetic code to the nucleotide sequence ofa nucleic acid molecule as defined in (a), (c) or (d).
 8. Thepolypeptide of claim 7, comprising: (a) an amino acid sequence which isa C-terminally or C- and N-terminally truncated version of the aminoacid sequence of SEQ ID NO: 24 and comprises maximal 50-95% of the aminoacid sequence of SEQ ID NO: 24; (b) a polypeptide encoded by a nucleicacid molecule encoding a polypeptide having an amino acid sequence whichis a C-terminally or C- and N-terminally truncated version of the aminoacid sequence of SEQ ID NO: 24 and comprises maximal 50-95% of the aminoacid sequence of SEQ ID NO: 24; or (c) a polypeptide having at least 80%identity to the polypeptide of (a) or (b), whereby said polypeptide isfunctional; wherein the function comprises the ability to transferGlcNAc-1P or a derivative thereof from UDP-GlcNAc or a derivativethereof onto an acceptor carbohydrate.
 9. The polypeptide of claim 7 or8, wherein the polypeptide comprises: (i) an amino acid sequence encodedby a nucleic acid molecule which comprises the nucleic acid sequence ofSEQ ID NO: 17, 20 or 21; or a nucleic acid sequence having at least 80%identity to the nucleic acid sequence of any one of SEQ ID NO: 17, 20 or21 and encoding a functional polypeptide; wherein the function comprisesthe ability to transfer GlcNAc-1P or a derivative thereof fromUDP-GlcNAc or a derivative thereof onto an acceptor carbohydrate; or(ii) the amino acid sequence of any one of SEQ ID NO: 25, 28 or 29; oran amino acid sequence having at least 80% identity to SEQ ID NO: 25, 28or 29 and being functional, wherein the function comprises the abilityto transfer GlcNAc-1P or a derivative thereof from UDP-GlcNAc or aderivative thereof onto an acceptor carbohydrate.
 10. The polypeptide ofany one of claims 7 to 9, wherein the function comprises the ability totransfer GlcNAc-1P or a derivative thereof from UDP-GlcNAc or aderivative thereof onto an acceptor carbohydrate; wherein at least 60%of the the produced polymers have a DP between 10 and 60 or an avDPbetween 15 and 20, when the ratio of donor carbohydrate to acceptorcarbohydrate is in the range of 10:1 to 1000:1.
 11. Nucleic acidmolecule encoding the polypeptide of any one of claims 7 to
 10. 12.Vector comprising the nucleic acid molecule of claim
 11. 13. Host cellcomprising the vector of claim
 12. 14. The method of any one of claims 1to 3 and 6, or the composition of any one of claims 4 to 6, wherein thetruncated version of the capsule polymerase of Neisseria meningitidisserogroup X is the polypeptide as defined in any one of claims 7 to 10.15. The method of any one of claims 1 to 3, 6 and 14, wherein at least60% of the produced capsular polysaccharides have a DP of 10 to 60 or anavDP of 15 to
 20. 16. The method of any one of claims 1, 3, 6, 14 and15, or the composition of any one of claims 4 to 6, 14 and 15, wherein(i) the ratio of donor carbohydrate to acceptor carbohydrate is a ratiofrom 20:1 to 80:1; and the capsule polymerase is either a polypeptidecomprising maximal 80% of the amino acid sequence of SEQ ID NO: 24 andcomprising the amino acid sequence of SEQ ID NO: 28, or a polypeptidecomprising the amino acid sequence of SEQ ID NO: 9; or (ii) the ratio ofdonor carbohydrate to acceptor carbohydrate is a ratio from 100:1 to400:1; and the capsule polymerase is a polypeptide comprising maximal68% of the amino acid sequence of SEQ ID NO: 24 and comprising the aminoacid sequence of SEQ ID NO:
 29. 17. The method of any one of claims 1 to3, 6 and 14 to 16, wherein said donor carbohydrate is activated orwherein said donor carbohydrate is activated during step (a).
 18. Themethod of claim 17, wherein said donor carbohydrate is activated duringstep (a) by contacting said donor carbohydrate with an activatingenzyme.
 19. The method of claim 18, wherein in step (a) said donorcarbohydrate is further contacted with PEP and/or at least oneactivating nucleotide.
 20. The composition of any one of claims 4 to 6,14 and 16, wherein said donor carbohydrate is activated.
 21. The methodof any one of claims 17 to 19 or the composition of claim 20, whereinsaid donor carbohydrate is activated by linkage to an activatingnucleotide.
 22. The method of claim 19 or 21 or the composition of claim21, wherein said activating nucleotide is CMP, CDP, CTP, UMP, UDP, TDP,AMP or UTP.
 23. The method of any one of claims 1 to 3, 6, 14 to 19, 21and 22 or the composition of any one of claims 4 to 6, 14, 16 and 20 to22, wherein the capsule polymerase is a truncated version of the capsulepolymerase of Neisseria meningitidis serogroup X and wherein at leastone donor carbohydrate is UDP-GlcNAc.
 24. The method of any one ofclaims 1 to 3, 6, 14 to 19 and 21 to 23 or the composition of any one ofclaims 4 to 6, 14, 16 and 20 to 23, wherein the capsule polymerase is atruncated version of the capsule polymerase of Neisseria meningitidisserogroup X and wherein at least one donor carbohydrate is GlcNAc-1-P.25. The method of any one of claims 1, 6, 14 to 19, 21 and 22 or thecomposition of any one of claims 4 to 6, 14, 16 and 20 to 22, whereinthe capsule polymerase is the capsule polymerase of Neisseriameningitidis serogroup A and wherein at least one donor carbohydrate isUDP-ManNAc.
 26. The method of any one of claims 1, 6, 14 to 19, 21, 22and 25 or the composition of any one of claims 4 to 6, 14, 16, 20 to 22and 25, wherein the capsule polymerase is the capsule polymerase ofNeisseria meningitidis serogroup A and wherein at least one donorcarbohydrate is UDP-GlcNAc.
 27. The method of claim 26, wherein in step(a) the capsule polymerase is further incubated with theUDP-GlcNAc-epimerase.
 28. The composition of claim 26, furthercomprising the UDP-GlcNAc-epimerase.
 29. The method of any one of claims1, 6, 14 to 19, 21, 22 and 25 to 27 or the composition of any one ofclaims 4 to 6, 14, 16, 29 to 22, 25, 26 and 28, wherein the capsulepolymerase is the capsule polymerase of Neisseria meningitidis serogroupA and wherein at least one donor carbohydrate is ManNAc-1-P.
 30. Themethod of any one of claims 1 to 3, 6, 14 to 19, 21 to 27 and 29 or thecomposition of any one of claims 4 to 6, 14, 16, 20 to 26, 28 and 29,wherein said acceptor carbohydrate is capsule polysaccharide ofNeisseria meningitidis serogroup A or X or a carbohydrate structurecontaining terminal GlcNAc residues.
 31. The method of claim 30 or thecomposition of claim 30, wherein the carbohydrate structure containingterminal GlcNAc-residues is hyaluronic acid, heparin sulphate, heparansulphate or protein-linked oligosaccharides.
 32. The method of any oneof claims 1 to 3, 6, 14 to 19, 21 to 27 and 29 or the composition of anyone of claims 4 to 6, 14, 16, 20 to 26, and 28-30, wherein at least 80%of the acceptor carbohydrates are polysaccharides with a DP of ≧4. 33.The method of any one of claims 1 to 3, 6, 14 to 19, 21 to 27 and 29 to32 or composition of any one of claims 4 to 6, 14, 16, 20 to 26 and 28to 32, wherein the acceptor carbohydrate is non-acetylated and/ordephosphorylated, preferably non-acetylated and dephosphorylated. 34.The method of any one of claims 1 to 3, 6, 14 to 19, 21 to 27 and 29 to33 or the composition of any one of claims 4 to 6, 14, 16, 20 to 26 and28 to 33, wherein the acceptor carbohydrate carries one or moreadditional functional groups at its reducing end.
 35. The method of anyone of claims 1 to 3, 6, 14 to 19, 21 to 27 and 29 to 31, and 33 or thecomposition of any one of claims 4 to 6, 14, 16, 20 to 26 and 28 to 31,and 33, wherein said acceptor carbohydrate is a dimer of ManNAc carryinga phosphodiester at the reducing end.
 36. The method of claim 35 or thecomposition of claim 35, wherein the phosphate group at the reducing endis extended with alkyl-azides, alkyl-amindes or sulfhydryl-groups. 37.The method of claim 35 or 36 or the composition of claim 35 or 36,wherein the acceptor carbohydrate is a disaccharide carrying adecyl-phosphate-ester at the reducing end.
 38. The method of any one ofclaims 1 to 3, 6, 14 to 19, 21 to 27 and 29 to 37 or the composition ofany one of claims 4 to 6, 14, 16, 20 to 26 and 28 to 37, wherein saidacceptor carbohydrate is purified.
 39. The method of any one of claims 1to 3, 6, 14 to 19, 21 to 27 and 29 to 38, further comprisingO-acetylation of the produced capsule polysaccharides.
 40. The method ofclaim 39, wherein the O-acetylation is performed by contacting theproduced capsule polysaccharides with an O-acetyltransferase.
 41. Thecomposition of any one of claims 4 to 6, 14, 16, 20 to 26 and 28 to 38,further comprising an O-acetyltransferase.
 42. The method of claim 40 orcomposition of claim 41, wherein the O-acetyltransferase is thepolypeptide of any one of (a) to (f): (a) a polypeptide comprising anamino acid sequence encoded by a nucleic acid molecule having thenucleic acid sequence of SEQ ID NO: 53; (b) a polypeptide comprising theamino acid sequence of SEQ ID NO: 54; (c) a polypeptide encoded by anucleic acid molecule encoding a polypeptide comprising the amino acidsequence of SEQ ID NO: 54 or of a functional fragment thereof, whereinthe function comprises the ability to transfer Acetyl-groups from thedonor Acetyl-Coenzyme A onto hydroxyl-groups of UDP-ManNAc or oligo- andpolymeric structures consisting of ManNAc-1-phosphate units linkedtogether by phosphodiester linkages; (d) a polypeptide comprising anamino acid sequence encoded by a nucleic acid molecule hybridizing understringent conditions to the complementary strand of a nucleic acidmolecule as defined in (a) or (c) and encoding a functional polypeptide;or a functional fragment thereof, wherein the function comprises theability to transfer Acetyl-groups from the donor Acetyl-Coenzyme A ontohydroxyl-groups of UDP-ManNAc or oligo- and polymeric structuresconsisting of ManNAc-1-phosphate units linked together by phosphodiesterlinkages; (e) a polypeptide having at least 80% identity to thepolypeptide of any one of (a) to (d), whereby said polypeptide isfunctional; or a functional fragment thereof, wherein the functioncomprises the ability to transfer Acetyl-groups from the donorAcetyl-Coenzyme A onto hydroxyl-groups of UDP-ManNAc or oligo- andpolymeric structures consisting of ManNAc-1-phosphate units linkedtogether by phosphodiester linkages; and (f) a polypeptide comprising anamino acid sequence encoded by a nucleic acid molecule being degenerateas a result of the genetic code to the nucleotide sequence of a nucleicacid molecule as defined in (a), (c), and (d).
 43. The method of any oneof claims 35 to 40 and 42 or the composition of any one of claims 35 to38 and 41, wherein the capsule polymerase is the capsule polymerase ofNeisseria meningitidis serogroup A.
 44. The method of claim 43, whereinthe produced capsule polysaccharides are O-acetylated in positions 3 and4 of ManNAc.
 45. The method of any one of claims 1 to 3, 6, 14 to 19, 21to 27, 29 to 39 and 41 to 44, further comprising covalently attachingthe produced capsular polysaccharides to a carrier molecule.
 46. Themethod of claim 45, wherein the carrier molecule is tetanus toxoidtetanus toxoid and CRM₁₉₇ an inactive form of diphteria-toxin.
 47. Themethod of any one of claims 1, 6, 14, 15, 17 to 19, 21 to 27, 29 to 39,and 41 to 46, wherein the ratio of donor carbohydrate to acceptorcarbohydrate is a ratio from 20:1 to 400:1.
 48. The method of any one ofclaims 3, 6, 14, 15, 17 to 19, 21 to 27, 29 to 39, and 41 to 46, whereinthe ratio of donor carbohydrate to acceptor carbohydrate is a ratio from200:1 to 1000:1.
 49. The composition of any one of claims 4 to 6, 14,16, 20 to 26, 28 to 38, and 41 to 43, wherein the ratio of donorcarbohydrate to acceptor carbohydrate is a ratio from 20:1 to 1000:1.50. The method of any one of claims 1 to 3, 6, 14 to 19, 21 to 27, 29 to39 and 41 to 48, wherein the capsule polymerase is immobilized on asolid phase.
 51. A capsular polysaccharide which has been produced bythe method of any one of claims 1 to 3, 6, 14 to 19, 21 to 27, 29 to 39,41 to 48 and
 50. 52. The capsular polysaccharide of claim 51 for use asa medicament.
 53. A vaccine comprising the capsular polysaccharide ofclaim 51, optionally further comprising a pharmaceutically acceptablecarrier.
 54. A method for producing a vaccine comprising the method ofany one of claims 1 to 3, 6, 14 to 19, 21 to 27, 29 to 39 and 41 to 48and
 50. 55. The capsular polysaccharide of claim 51 or 52 or the vaccineof claim 53 for use in vaccination of a subject.
 56. The capsularpolysaccharide of claim 55 or the vaccine of claim 55, wherein thesubject is a human being.
 57. The capsular polysaccharide of claim 555or 56 or the vaccine of claim 55 or 56, wherein the vaccination isagainst meningococcal meningitidis caused by Neisseria meningitidisserogroup A or X.